Human timp-1 antibodies

ABSTRACT

Human antibodies that bind to TIMP-1 can be used as reagents to diagnose and treat disorders in which TIMP-1 is elevated, such as liver fibrosis, alcoholic liver disease, cardiac fibrosis, acute coronary syndrome, lupus nephritis, glomerulosclerotic renal disease, benign prostate hypertrophy, colon cancer, lung cancer, and idiopathic pulmonary fibrosis.

Under 35 USC §120, this application is a continuation application ofU.S. patent application Ser. No. 13/174,510, filed Jun. 30, 2011, whichis a Divisional application of U.S. patent application Ser. No.12/195,286, filed Aug. 20, 2008, now issued as U.S. Pat. No. 7,993,849,which is a continuation application of U.S. patent application Ser. No.11/504,527, filed Aug. 14, 2006, now issued as U.S. Pat. No. 7,432,364,which is a continuation application of U.S. patent application Ser. No.10/128,520, filed Apr. 24, 2002, now issued as U.S. Pat. No. 7,091,323,which claims the benefit under 35 USC §119(e) to U.S. Patent ApplicationSer. No. 60/285,683 filed Apr. 24, 2001. The disclosure of each of theprior applications is considered part of and is incorporated byreference in the disclosure of this application.

This application incorporates by reference the sequence listing entitled“Human TIMP-1 Antibodies,” which is part of the application.

FIELD OF THE INVENTION

The invention relates to TIMP-1-binding human antibodies.

BACKGROUND OF THE INVENTION

Tissue inhibitors of metalloproteases (TIMPs) inhibit metalloproteases,a family of endopeptide hydrolases. Metalloproteases are secreted byconnective tissue and hematopoietic cells, use Zn²⁺ or Ca²⁺ forcatalysis, and may be inactivated by metal chelators as well as TIMPmolecules. Matrix metalloproteases (MMPs) participate in a variety ofbiologically important processes, including the degradation of manystructural components of tissues, particularly the extracellular matrix(ECM).

Degradation of extracellular matrix tissue is desirable in processeswhere destruction of existing tissues is necessary, e.g., in embryoimplantation (Reponen et al., Dev. Dyn. 202, 388-96, 1995),embryogenesis, and tissue remodeling. Imbalance between synthesis anddegradation of matrix proteins, however, can result in diseases such asliver fibrosis (Iredale et al., Hepatology 24, 176-84, 1996). Thisimbalance can occur, for example, if levels of TIMPs are increased.Disorders in which TIMP-1 levels of increased include, for example,liver fibrosis, alcoholic liver disease, cardiac fibrosis, acutecoronary syndrome, lupus nephritis, glomerulosclerotic renal disease,idiopathic pulmonary fibrosis, benign prostate hypertrophy, lung cancer,and colon cancer. See, e.g., Inokubo et al., Am. Heart J. 141, 211-17,2001; Ylisirnio et al., Anticancer Res. 20, 1311-16, 2000;Holten-Andersen et al., Clin. Cancer Res. 6, 4292-99, 2000;Holten-Andersen et al., Br. J. Cancer 80, 495-503, 1999; Peterson etal., Cardiovascular Res. 46, 307-15, 2000; Arthur et al., Alcoholism:Clinical and Experimental Res. 23, 840-43, 1999; Iredale et al.,Hepatol. 24, 176-84, 1996.

There is a need in the art for reagents and methods of inhibiting TIMP-1activity, which can be used to provide therapeutic effects.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide reagents and methodsof inhibiting TIMP-1 activity. This and other objects of the inventionare provided by one or more of the embodiments described below.

One embodiment of the invention is a purified preparation of a humanantibody, wherein the antibody binds to a tissue inhibitor ofmetalloprotease-1 (TIMP-1) and neutralizes a matrix metalloprotease(MMP)-inhibiting activity of the TIMP-1.

Another embodiment of the invention is a purified preparation of a firsthuman antibody which comprises a VHCDR3 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOS:1-43 and 360.

Still another embodiment of the invention is a purified preparation of afirst human antibody which comprises a VLCDR3 region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOS:44-86 and365-379.

Yet another embodiment of the invention is a purified preparation of afirst human antibody which has TIMP-1 binding and MMP-inhibitingactivity characteristics of a second human antibody. The second antibodycomprises a VHCDR3 and VLCDR3 amino acid sequence pair selected from thegroup consisting of SEQ ID NOS:1 and 44, SEQ ID NOS:2 and 45, SEQ IDNO:3 and 46, SEQ ID NOS:4 and 47, SEQ ID NOS:5 and 48, SEQ ID NOS:6 and49, SEQ ID NOS:7 and 50, SEQ ID NOS:3 and 44, SEQ ID NOS:3 and 45, SEQID NOS:3 and 47, SEQ ID NOS:3 and 48, SEQ ID NOS:3 and 49, SEQ ID NOS:3and 50, SEQ ID NOS:7 and 44, SEQ ID NOS:7 and 45, SEQ ID NOS:7 and 47,SEQ ID NOS:7 and 48, SEQ ID NOS:8 and 51, SEQ ID NOS:9 and 52, SEQ IDNOS:10 and 53, SEQ ID NOS:11 and 54, SEQ ID NOS:12 and 55, SEQ ID NOS:13and 56, SEQ ID NOS:14 and 57, SEQ ID NOS:15 and 58, SEQ ID NOS:16 and59, SEQ ID NOS:17 and 60, SEQ ID NOS:18 and 61, SEQ ID NOS:19 and 62,SEQ ID NOS:20 and 63, SEQ ID NOS:21 and 64, SEQ ID NOS:22 and 65, SEQ IDNOS:23 and 66, SEQ ID NOS:24 and 67, SEQ ID NOS:25 and 68, SEQ ID NOS:26and 69, SEQ ID NOS: 27 and 70, SEQ ID NOS:28 and 71, SEQ ID NOS:29 and72, SEQ ID NOS:30 and 73, SEQ ID NOS:31 and 74, SEQ ID NOS:32 and 75,SEQ ID NOS:33 and 76, SEQ ID NOS:34 and 77, SEQ ID NOS:35 and 78, SEQ IDNOS:36 and 79, SEQ ID NOS:37 and 80, SEQ ID NOS:38 and 81, SEQ ID NOS:39and 82, SEQ ID NOS:40 and 83, SEQ ID NOS:41 and 84, SEQ ID NOS:42 and85, SEQ ID NOS:43 and 86, SEQ ID NOS:3 and 48, SEQ ID NOS:360 and 48,SEQ ID NOS:3 and 365, SEQ ID NOS:16 and 59, SEQ ID NOS:18 and 61, SEQ IDNOS:34 and 77, SEQ ID NOS:34 and 379, SEQ ID NOS:18 and 376, SEQ IDNOS:18 and 377, and SEQ ID NOS:18 and 378.

Even another embodiment of the invention is a purified preparation of ahuman antibody comprising a VHCDR3 and VLCDR3 amino acid sequence pairselected from the group consisting of SEQ ID NOS:1 and 44, SEQ ID NOS:2and 45, SEQ ID NO:3 and 46, SEQ ID NOS:4 and 47, SEQ ID NOS:5 and 48,SEQ ID NOS:6 and 49, SEQ ID NOS:7 and 50, SEQ ID NOS:3 and 44, SEQ IDNOS:3 and 45, SEQ ID NOS:3 and 47, SEQ ID NOS:3 and 48, SEQ ID NOS:3 and49, SEQ ID NOS:3 and 50, SEQ ID NOS:7 and 44, SEQ ID NOS:7 and 45, SEQID NOS:7 and 47, SEQ ID NOS:7 and 48, SEQ ID NOS:8 and 51, SEQ ID NOS:9and 52, SEQ ID NOS:10 and 53, SEQ ID NOS:11 and 54, SEQ ID NOS:12 and55, SEQ ID NOS:13 and 56, SEQ ID NOS:14 and 57, SEQ ID NOS:15 and 58,SEQ ID NOS:16 and 59, SEQ ID NOS:17 and 60, SEQ ID NOS:18 and 61, SEQ IDNOS:19 and 62, SEQ ID NOS:20 and 63, SEQ ID NOS:21 and 64, SEQ ID NOS:22and 65, SEQ ID NOS:23 and 66, SEQ ID NOS:24 and 67, SEQ ID NOS:25 and68, SEQ ID NOS:26 and 69, SEQ ID NOS: 27 and 70, SEQ ID NOS:28 and 71,SEQ ID NOS:29 and 72, SEQ ID NOS:30 and 73, SEQ ID NOS:31 and 74, SEQ IDNOS:32 and 75, SEQ ID NOS:33 and 76, SEQ ID NOS:34 and 77, SEQ ID NOS:35and 78, SEQ ID NOS:36 and 79, SEQ ID NOS:37 and 80, SEQ ID NOS:38 and81, SEQ ID NOS:39 and 82, SEQ ID NOS:40 and 83, SEQ ID NOS:41 and 84,SEQ ID NOS:42 and 85, SEQ ID NOS:43 and 86, SEQ ID NOS:3 and 48, SEQ IDNOS:360 and 48, SEQ ID NOS:3 and 365, SEQ ID NOS:16 and 59, SEQ IDNOS:18 and 61, SEQ ID NOS:34 and 77, SEQ ID NOS:34 and 379, SEQ IDNOS:18 and 376, SEQ ID NOS:18 and 377, and SEQ ID NOS:18 and 378.

A further embodiment of the invention is a purified preparation of ahuman antibody which comprises a heavy chain and a light chain aminoacid pair selected from the group consisting of SEQ ID NOS:140 and 97,SEQ ID NOS:141 and 98, SEQ ID NOS:142 and 99, SEQ ID NOS:143 and 100,SEQ ID NOS:144 and 101, SEQ ID NOS:145 and 102, SEQ ID NOS:146 and 103,SEQ ID NOS:142 and 97, SEQ ID NOS:142 and 98, SEQ ID NOS:142 and 100,SEQ ID NOS:142 and 101, SEQ ID NOS:142 and 102, SEQ ID NOS:142 and 103,SEQ ID NOS:146 and 97, SEQ ID NOS:146 and 98, SEQ ID NO:146 and 100, SEQID NOS:146 and 101, SEQ ID NOS:148 and 104, SEQ ID NOS:148 and 105, SEQID NOS:149 and 106, SEQ ID NOS:150 and 107, SEQ ID NOS:151 and 108, SEQID NOS:152 and 109, SEQ ID NOS:153 and 110, SEQ ID NOS:154 and 111, SEQID NOS:155 and 112, SEQ ID NOS:156 and 113, SEQ ID NOS:157 and 114, SEQID NOS:158 and 115, SEQ ID NOS:159 and 116, SEQ ID NOS:160 and 117, SEQID NOS:161 and 118, SEQ ID NOS:162 and 119, SEQ ID NOS:163 and 120, SEQID NOS:164 and 121, SEQ ID NOS:165 and 122, SEQ ID NOS:166 and 123, SEQID NOS:167 and 124, SEQ ID NOS:168 and 125, SEQ ID NOS:169 and 126, SEQID NOS:170 and 127, SEQ ID NOS:171 and 128, SEQ ID NOS:172 and 129, SEQID NOS:173 and 130, SEQ ID NOS:174 and 131, SEQ ID NOS:175 and 132, SEQID NOS:176 and 133, SEQ ID NOS:177 and 134, SEQ ID NOS:178 and 135, SEQID NOS:179 and 136, SEQ ID NOS:180 and 137, SEQ ID NOS:181 and 138, andSEQ ID NOS:182 and 139.

Another embodiment of the invention is a pharmaceutical compositioncomprising a human antibody and a pharmaceutically acceptable carrier.The human antibody (1) binds to a TIMP-1 and (2) neutralizes anMMP-inhibiting activity of the TIMP-1.

Yet another embodiment of the invention is a purified polynucleotidewhich encodes a human antibody comprising a VHCDR3 region whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:1-43 and 360. The human antibody (1) binds to a TIMP-1 and(2) neutralizes an MMP-inhibiting activity of the TIMP-1.

Even another embodiment of the invention is a purified polynucleotidewhich encodes a human antibody comprising a VLCDR3 region whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:44-86 and 365-379. The human antibody (1) binds to a TIMP-1and (2) neutralizes an MMP-inhibiting activity of the TIMP-1.

Still another embodiment of the invention is an expression vectorcomprising a polynucleotide which encodes a human antibody comprising aVHCDR3 region which comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOS:1-43 and 360. The human antibody (1)binds to a TIMP-1 and (2) neutralizes an MMP-inhibiting activity of theTIMP-1.

A further embodiment of the invention is an expression vector comprisinga polynucleotide which encodes a human antibody comprising a VHCDR3region which comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS:1-43 and 360. The human antibody (1) binds to aTIMP-1 and (2) neutralizes an MMP-inhibiting activity of the TIMP-1. TheVHCDR3 region is encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NOS:227-269.

Another embodiment of the invention is an expression vector comprising apolynucleotide which encodes a human antibody comprising a VLCDR3 regionwhich comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS:44-86 and 365-379. The human antibody (1) bindsto a TIMP-1 and (2) neutralizes an MMP-inhibiting activity of theTIMP-1.

Yet another embodiment of the invention is an expression vectorcomprising a polynucleotide which encodes a human antibody comprising aVLCDR3 region which comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOS:44-86 and 365-379. The human antibody (1)binds to a TIMP-1 and (2) neutralizes an MMP-inhibiting activity of theTIMP-1. The VLCDR3 region is encoded by a nucleotide sequence selectedfrom the group consisting of SEQ ID NOS:184-226.

Still another embodiment of the invention is an expression vectorcomprising a polynucleotide which encodes a human antibody comprising aVHCDR3 region which comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOS:1-43 and 360. The human antibody (1)binds to a TIMP-1 and (2) neutralizes an MMP-inhibiting activity of theTIMP-1. The human antibody comprises a heavy chain having an amino acidsequence selected from the group consisting of SEQ ID NOS:140-182.

Even another embodiment of the invention is an expression vectorcomprising a polynucleotide which encodes a human antibody comprising aVHCDR3 region which comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOS:1-43 and 360. The human antibody (1)binds to a TIMP-1 and (2) neutralizes an MMP-inhibiting activity of theTIMP-1. The human antibody comprises a heavy chain having an amino acidsequence selected from the group consisting of SEQ ID NOS:140-182. Theheavy chain is encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NOS:269-311.

A further embodiment of the invention is an expression vector comprisinga polynucleotide which encodes a human antibody comprising a VLCDR3region which comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS:44-86 and 365-379. The human antibody (1) bindsto a TIMP-1 and (2) neutralizes an MMP-inhibiting activity of theTIMP-1. The human antibody comprises a light chain having an amino acidsequence selected from the group consisting of SEQ ID NOS:97-139.

Another embodiment of the invention is an expression vector comprising apolynucleotide which encodes a human antibody comprising a VLCDR3 regionwhich comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS:44-86 and 365-379. The human antibody (1) bindsto a TIMP-1 and (2) neutralizes an MMP-inhibiting activity of theTIMP-1. The human antibody comprises a light chain having an amino acidsequence selected from the group consisting of SEQ ID NOS:97-139. Thelight chain is encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NOS:312-354.

Yet another embodiment of the invention is a host cell comprising anexpression vector. The expression vector comprises a polynucleotidewhich encodes a human antibody comprising a VHCDR3 region whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:1-43 and 360, wherein the human antibody (1) binds to aTIMP-1 and (2) neutralizes an MMP-inhibiting activity of the TIMP-1.

Yet another embodiment of the invention is a host cell comprising anexpression vector. The expression vector comprises a polynucleotidewhich encodes a human antibody comprising a VHCDR3 region whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:1-43 and 360, wherein the human antibody (1) binds to aTIMP-1 and (2) neutralizes an MMP-inhibiting activity of the TIMP-1. TheVHCDR3 region is encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NOS:227-269.

Still another embodiment of the invention is a host cell comprising anexpression vector. The expression vector comprises a polynucleotidewhich encodes a human antibody comprising a VLCDR3 region whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:44-86 and 365-379. The human antibody (1) binds to a TIMP-1and (2) neutralizes an MMP-inhibiting activity of the TIMP-1.

A further embodiment of the invention is a host cell comprising anexpression vector. The expression vector comprises a polynucleotidewhich encodes a human antibody comprising a VLCDR3 region whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:44-86 and 365-379. The human antibody (1) binds to a TIMP-1and (2) neutralizes an MMP-inhibiting activity of the TIMP-1. The VLCDR3region is encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NOS:184-226.

Another embodiment of the invention is a host cell comprising anexpression vector. The expression vector comprises a polynucleotidewhich encodes a human antibody comprising a VHCDR3 region whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:1-43 and 360, wherein the human antibody (1) binds to aTIMP-1 and (2) neutralizes an MMP-inhibiting activity of the TIMP-1. Thehuman antibody comprises a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NOS:140-182.

Still another embodiment of the invention is a host cell comprising anexpression vector. The expression vector comprises a polynucleotidewhich encodes a human antibody comprising a VHCDR3 region whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:1-43 and 360, wherein the human antibody (1) binds to aTIMP-1 and (2) neutralizes an MMP-inhibiting activity of the TIMP-1. Thehuman antibody comprises a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NOS:140-182. The heavychain is encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NOS:269-311.

Yet another embodiment of the invention is a host cell comprising anexpression vector. The expression vector comprises a polynucleotidewhich encodes a human antibody comprising a VLCDR3 region whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:44-86 and 365-379. The human antibody (1) binds to a TIMP-1and (2) neutralizes an MMP-inhibiting activity of the TIMP-1. The humanantibody comprises a light chain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOS:97-139.

Even another embodiment of the invention is a host cell comprising anexpression vector. The expression vector comprises a polynucleotidewhich encodes a human antibody comprising a VLCDR3 region whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:44-86 and 365-379. The human antibody (1) binds to a TIMP-1and (2) neutralizes an MMP-inhibiting activity of the TIMP-1. The humanantibody comprises a light chain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOS:97-139. The light chain isencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NOS:312-354.

A further embodiment of the invention is a method of making a humanantibody. The host cell of claim 43 is cultured under conditions wherebythe antibody is expressed. The human antibody is purified from the hostcell culture.

Another embodiment of the invention is a method of decreasing anMMP-inhibiting activity of a TIMP-1. The TIMP-1 is contacted with ahuman antibody that binds to the TIMP-1. The MMP-inhibiting activity ofthe TIMP-1 is decreased relative to MMP-inhibiting activity of theTIMP-1 in the absence of the antibody.

Still another embodiment of the invention is a method of amelioratingsymptoms of a disorder in which TIMP-1 is elevated. An effective amountof a human antibody which neutralizes an MMP-inhibiting activity of theTIMP-1 is administered to a patient having the disorder. Symptoms of thedisorder are thereby ameliorated.

A further embodiment of the invention is a method of detecting a TIMP-1in a test preparation. The test preparation is contacted with a humanantibody that specifically binds to the TIMP-1. The test preparation isassayed for the presence of an antibody-TIMP-1 complex.

Even another embodiment of the invention is a method to aid indiagnosing a disorder in which a TIMP-1 level is elevated. A sample froma patient suspected of having the disorder is contacted with a humanantibody that binds to TIMP-1. The sample is assayed for the presence ofan antibody-TIMP-1 complex. Detection of an amount of the complex whichis greater than an amount of the complex in a normal sample identifiesthe patient as likely to have the disorder.

The invention thus provides human antibodies which bind to TIMP-1 andneutralize MMP-inhibiting activity of TIMP-1. These antibodies can beused, inter alia, in diagnostic and therapeutic methods.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Protein sequences encoded by the HuCAL® V_(H) and V_(L) Fabmaster genes. Seven V_(H) and V_(L) sequences are aligned, and theapproximate location of restriction endonuclease sites introduced intothe corresponding DNA sequences are indicated. The numbering isaccording to VBASE except for the gap in V1 position 9. In VBASE the gapis set at position 10. See also Chothia et al. (1992) J. Mol. Biol. 227,776-798, Tomlinson et al. (1995) EMBO J. 14, 4628-4638 and Williams etal. (1996) J. Mol. Biol. 264, 220-232).

FIGS. 2A-2C. Nucleotide sequences of the HuCAL® V_(H) and V_(L) Fabmaster genes.

FIG. 3. Fab display vector pMORPH® 18 Fab 1.

FIG. 4. Vector map of pMORPH® x9Fab1_FS.

FIG. 5. Sequence comparison between human and rat TIMP-1. Sequenceregions in bold were used for peptide synthesis. Residues that makestronger direct contacts with MMP-3 are italicized, and residues thatmake weaker direct contacts with MMP-3 are underlined (Gomis-Ruth etal., 1997).

FIG. 6. Activity of MS-BW-3 in human TIMP-1/MMP-1 assay. Antibody Fabfragments were diluted in triplicate to the indicated concentrations inassay buffer containing 0.05% BSA. After addition of TIMP (final conc.1.2 nM), MMP (final conc. 1.2 nM), and peptide substrate (final conc. 50μM) and incubation for 1-3 h at 37° C. fluorescence at Ex320 nm/Em 430nm was measured. IC₅₀ was calculated as outlined in material and methodssection, using 100% MMP-1 activity (in absence of TIMP-1) and 27% MMP-1activity (in absence of antibody) as reference values.

FIG. 7. Activity of MS-BW-44 in human TIMP-1/MMP-1 assay. Antibody Fabfragments were diluted in triplicate to the indicated concentrations inassay buffer containing 0.05% BSA. After addition of TIMP (final conc.1.2 nM), MMP (final conc. 1.2 nM), and peptide substrate (final conc. 50μM) and incubation for 1-3 h at 37° C. fluorescence at Ex320 nm/Em 430nm was measured. IC₅₀ was calculated as outlined in material and methodssection, using 100% MMP-1 activity (in absence of TIMP-1) and 25% MMP-1activity (in absence of antibody) as reference values.

FIG. 8. Activity of MS-BW-44, -44-2, 44-6 in human TIMP-1/MMP-1 assay.Fab antibody fragments were diluted in triplicate to the indicatedconcentrations in assay buffer containing 0.05% BSA. After addition ofTIMP (final conc. 0.4 nM), MMP (final conc. 0.4 nM) and peptidesubstrate (final conc. 50 μM) and incubation for 7 h at 37° C.fluorescence at Ex320 nm/Em 430 nm was measured. IC₅₀ was calculated asoutlined in material and methods section, using 100% MMP-1 activity (inabsence of TIMP-1) and 55% MMP-1 activity (in absence of antibody) asreference values.

FIG. 9. Activity of MS-BW-44, -44-2-4, 44-6-1 in human TIMP-1/MMP-1assay. Antibody Fab fragments were diluted in triplicate to theindicated concentrations in assay buffer containing 0.05% BSA. Afteraddition of TIMP (final conc. 0.4 nM), MMP (final conc. 0.4 nM), andpeptide substrate (final conc. 50 μM) and incubation for 7 h at 37° C.fluorescence at Ex320 nm/Em 430 nm was measured. IC₅₀ was calculated asoutlined in material and methods section, using 100% MMP-1 activity (inabsence of TIMP-1) and 50% MMP-1 activity (in absence of antibody) asreference values.

FIG. 10. Binding of Fab fragments to human TIMP-1, -2, -3 and -4.TIMP-1, -2, -3, -4 proteins were immobilized on an ELISA plate, andbinding of purified Fab fragments was measured by incubation withalkaline phosphatase conjugated anti-Fab antibody (Dianova) followed bydevelopment with Attophos substrate (Roche) and measurement at Ex405nm/Em535 nm.

FIG. 11. Activity of MS-BW-14, -17, -54 in rat TIMP-1/MMP-13 assay.Antibody Fab fragments were diluted in triplicate to the indicatedconcentrations in assay buffer containing 0.05% BSA. After addition ofTIMP (final conc. 1.2 nM), MMP (final conc. 1.2 nM), and peptidesubstrate (to final conc. 50 μM) and incubation for 1-3 h at 37° C.fluorescence at Ex320 nm/Em 430 nm was measured. IC₅₀ was calculated asoutlined in material and methods section, using 100% MMP-13 (in absenceof TIMP-1) activity and 20% MMP-13 activity (in absence of antibody) asreference values.

FIG. 12. Activity of MS-BW-14 Fab and IgG₁ and MS-BW-3 IgG₁ in ratTIMP-1/MMP-13 assay. Antibodies were diluted in triplicate to theindicated concentrations in assay buffer containing 0.05% BSA. Afteraddition of TIMP (final conc. 1.2 nM), MMP (final conc. 1.2 nM) andpeptide substrate (to final conc. 50 μM) and incubation for 1-3 h at 37°C., fluorescence at Ex320 nm/Em 430 nm was measured. IC₅₀ was calculatedas outlined in material and methods section, using 100% MMP-13 activity(in absence of TIMP-1) and 30% MMP-13 activity (in absence of antibody)as reference values.

FIG. 13. Activity of MS-BW-17-1 Fab and IgG₁ in rat TIMP-1/MMP-13 assay.Fab antibody fragments were diluted in triplicate to the indicatedconcentrations in assay buffer containing 0.05% BSA. After addition ofTIMP (final conc. 1.2 nM), MMP (final conc. 1.2 nM) and peptidesubstrate (to final conc. 50 μM) and incubation for 1-3 h at 37° C.fluorescence at Ex320 nm/Em 430 nm was measured. IC₅₀ was calculated asoutlined in material and methods section, using 100% MMP-13 activity (inabsence of TIMP-1) and 15% MMP-13 activity (in absence of antibody) asreference values.

FIG. 14. Effect of the inhibitory effect of MS-BW-17-1 TIMP-1 antibodyon bleomycin-induced lung fibrotic collagen.

FIG. 15. Effect of anti-TIMP-1 antibody on fibrotic collagen as stainedby Sirus Red in carbon tetrachloride-induced rat liver fibrosis model.Sirius Red-stained area as percent of total field in carbontetrachloride-treated rats treated with PBS, control antibody, andMS-BW-14 anti-TIMP-1 antibody.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides human antibodies that bind to TIMP-1. Theseantibodies are useful for a variety of therapeutic and diagnosticpurposes.

Characteristics of Human TIMP-1 Antibodies

“Antibody” as used herein includes intact immunoglobulin molecules(e.g., IgG₁, IgG_(2a), IgG_(2b), IgG₃, IgM, IgD, IgE, IgA), as well asfragments thereof, such as Fab, F(ab′)2, scFv, and Fv, which are capableof specific binding to an epitope of a human and/or rat TIMP-1 protein.Antibodies that specifically bind to TIMP-1 provide a detection signalat least 5-, 10-, or 20-fold higher than a detection signal providedwith other proteins when used in an immunochemical assay. Preferably,antibodies that specifically bind to human and/or rat TIMP-1 do notdetect other proteins in immunochemical assays and can immunoprecipitatethe TIMP-1 from solution.

The K_(d) of human antibody binding to TIMP-1 can be assayed using anymethod known in the art, including technologies such as real-timeBimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal.Chem. 63, 2338-45, 1991, and Szabo et al., Curr. Opin. Struct. Biol. 5,699-705, 1995). BIA is a technology for studying biospecificinteractions in real time, without labeling any of the interactants(e.g., BIAcore™) Changes in the optical phenomenon surface plasmonresonance (SPR) can be used as an indication of real-time reactionsbetween biological molecules.

In a BIAcore™ assay, some human antibodies of the invention specificallybind to human TIMP-1 with a K_(d) of about 0.1 nM to about 10 μM, about2 nM to about 1 μM, about 2 nM to about 200 nM, about 2 nM to about 150nM, about 50 nM to about 100 nM, about 0.2 nM to about 13 nM, about 0.2nM to about 0.5 nM, about 2 nM to about 13 nM, and about 0.5 nM to about2 nM. More preferred human antibodies specifically bind to human TIMP-1with a K_(d) selected from the group consisting of about 0.2 nM, about0.3 nM, about 0.5 M, about 0.6 nM, about 2 nM, about 7 nM, about 10 nM,about 11 nM, and about 13 nM.

Other human antibodies of the invention specifically bind to rat TIMP-1with a K_(d) of about 0.1 nM to about 10 μM, about 2 nM to about 1 μM,about 2 nM to about 200 nM, about 2 nM to about 150 nM, about 50 nM toabout 100 nM, about 1.3 nM to about 13 nM, about 1.8 nM to about 10 nM,about 2 nM to about 9 nM, about 1.3 nM to about 9 nM, and about 2 nM toabout 10 nM. Preferred K_(d) s range from about 0.8 nM, about 1 nM,about 1.3 nM, about 1.9 nM, about 2 nM, about 3 nM, about 9 nM, about 10nM, about 13 nM, about 14 nM, and about 15 nM.

Preferably, antibodies of the invention neutralize an MMP-inhibitingactivity of the TIMP-1. The MMP can be, for example, MMP-1, MMP-2,MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-19,MMP-20 or MMP-23.

IC₅₀ for neutralizing MMP-inhibiting activity of TIMP-1 can be measuredby any means known in the art. Preferably, IC₅₀ is determined using thehigh throughput fluorogenic assay described in Bickett et al., Anal.Biochem. 212, 58-64, 1993. In a typical fluorogenic assay, the IC₅₀ of ahuman antibody for neutralizing human TIMP-1 MMP-inhibiting activityranges from about 0.1 nM to about 200 nM, about 1 nM to about 100 nM,about 2 nM to about 50 nM, about 5 nM to about 25 nM, about 10 nM toabout 15 nM, about 0.2 nM to about 11 nM, about 0.2 nM to about 4 nM,and about 4 nM to about 11 nM. The IC₅₀ for neutralizing human TIMP-1MMP-inhibiting activity of some human antibodies is about 0.2 nM, about0.3 nM, about 0.4 nM, about 4 nM, about 7 nM, about 9 nM, and about 11nM.

A typical IC₅₀ for neutralizing rat TIMP-1 MMP-inhibiting activityranges from about 0.1 nM to about 300 nM, about 1 nM to about 100 nM,about 2 nM to about 50 nM, about 5 nM to about 25 nM, about 10 nM toabout 15 nM, about 1.1 nM to about 14 nM, about 1.6 nM to about 11 nM,about 3 nM to about 7 nM, about 1.1 nM to about 7 nM, about 1.1 nM toabout 11 nM, about 3 nM to about 11 nM, and about 3 nM to about 14 nM.The IC₅₀ for neutralizing rat TIMP-1 MMP-inhibiting activity of somehuman antibodies is about 1.1 nM, about 1.6 nM, about 3 nM, about 7 nM,about 11 nM, about 14 nM, about 19 nM, about 20 nM, about 30 nM, andabout 100 nM.

Preferred human antibodies of the invention are those for which theK_(d) for binding to TIMP-1 and the IC₅₀ for neutralizing theMMP-inhibiting activity of the TIMP-1 are approximately equal.

A number of human antibodies having the TIMP-1 binding andMMP-inhibiting activity neutralizing characteristics described abovehave been identified by screening the MorphoSys HuCAL® Fab 1 library.The CDR cassettes assembled for the HuCAL® library were designed toachieve a length distribution ranging from 5 to 28 amino acid residues,covering the stretch from position 95 to 102. Knappik et al., J. Mol.Biol. 296, 57-86, 2000. Some clones, however, had shorter VHCDR3regions. In fact, it is a striking feature of anti-human TIMP-1 humanantibodies identified from this library that they all exhibit thecombination VH312 and a relatively short VHCDR3 region, typically fouramino acids.

In some embodiments of the invention, the VHCDR3 region of a humanantibody has an amino acid sequence shown in SEQ ID NOS:1-43. In otherembodiments of the invention, the VLCDR3 region of a human antibody hasan amino acid sequence shown in SEQ ID NOS:44-86. See Tables 2, 3, and7. Human antibodies which have TIMP-1 binding and MMP-inhibitingactivity neutralizing characteristics of antibodies such as thosedescribed above and in Tables 2, 3, and 7 also are human antibodies ofthe invention.

Obtaining Human Antibodies

Human antibodies with the TIMP-1 binding and MMP-activity neutralizingcharacteristics described above can be identified from the MorphoSysHuCAL® library as follows. Human or rat TIMP-1, for example, is coatedon a microtiter plate and incubated with the MorphoSys HuCAL® Fab phagelibrary (see Example 1, below). Those phage-linked Fabs not binding toTIMP-1 can be washed away from the plate, leaving only phage whichtightly bind to TIMP-1. The bound phage can be eluted, for example, by achange in pH or by elution with E. coli and amplified by infection of E.coli hosts. This panning process can be repeated once or twice to enrichfor a population of antibodies that tightly bind to TIMP-1. The Fabsfrom the enriched pool are then expressed, purified, and screened in anELISA assay. The identified hits are then screened in the enzymaticassay described in Bickett et al., 1993, and Bodden et al., 1994. ThoseFabs that lead to the degradation of the peptide are likely the oneswhich bind to TIMP-1, thereby blocking its interaction to MMP-1.

The initial panning of the HuCAL® Fab 1 library also can be performedwith TIMP-1 as the antigen in round one, followed in round 2 by TIMP-1peptides fused to carrier proteins, such as BSA or transferrin, and inround 3 by TIMP-1 again. Human TIMP-1 peptides which can be used forpanning include human TIMP-1 residues 2-12 (TCVPPHPQTAF, SEQ ID NO:87;CTSVPPHPQTAF, SEQ ID NO:88; STCVPPHPQTAF, SEQ ID NO:89; STSVPPHPQTAFC,SEQ ID NO:90), 28-36 (CEVNQTTLYQ, SEQ ID NO:91), 64-75 (PAMESVCGYFHR,SEQ ID NO:92), 64-79 (PAMESVCGYFHRSHNR, SEQ ID NO:93; CPAMESVSGYFHRSHNR,SEQ ID NO:94; PAMESVSGYFHRSHNRC, SEQ ID NO:95), and 145-157(CLWTDQLLQGSE, SEQ ID NO:96). These peptide sequences are selected fromregions of human TIMP-1 that are predicted to interact with MMPs. SeeGomis-Ruth et al., Nature 389, 77-81, 1997. Directing Fabs toward theMMP-interacting region of human TIMP-1 in round 2 should increase thechance of identifying Fabs that can block the ability of human TIMP-1 toinhibit human MMP-1 activity.

Another method that can be used to improve the likelihood of isolatingneutralizing Fabs is the panning on human TIMP-1 and eluting the bindingFabs with human MMP-1. This strategy should yield higher affinityantibodies than would otherwise be obtained.

Details of the screening process are described in the specific examples,below. Other selection methods for highly active specific antibodies orantibody fragments can be envisioned by those skilled in the art andused to identify human TIMP-1 antibodies.

Human antibodies with the characteristics described above also can bepurified from any cell that expresses the antibodies, including hostcells that have been transfected with antibody-encoding expressionconstructs. The host cells are cultured under conditions whereby thehuman antibodies are expressed. A purified human antibody is separatedfrom other compounds that normally associate with the antibody in thecell, such as certain proteins, carbohydrates, or lipids, using methodswell known in the art. Such methods include, but are not limited to,size exclusion chromatography, ammonium sulfate fractionation, ionexchange chromatography, affinity chromatography, and preparative gelelectrophoresis. A preparation of purified human antibodies is at least80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purityof the preparations can be assessed by any means known in the art, suchas SDS-polyacrylamide gel electrophoresis. A preparation of purifiedhuman antibodies of the invention can contain more than one type ofhuman antibody with the TIMP-1 binding and neutralizing characteristicsdescribed above.

Alternatively, human antibodies can be produced using chemical methodsto synthesize its amino acid sequence, such as by direct peptidesynthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc.85, 2149-54, 1963; Roberge et al., Science 269, 202-04, 1995). Proteinsynthesis can be performed using manual techniques or by automation.Automated synthesis can be achieved, for example, using AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally,fragments of human antibodies can be separately synthesized and combinedusing chemical methods to produce a full-length molecule.

The newly synthesized molecules can be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton,PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., NewYork, N.Y., 1983). The composition of a synthetic polypeptide can beconfirmed by amino acid analysis or sequencing (e.g., using Edmandegradation).

Assessment of Therapeutic Utility of Human Antibodies

To assess the ability of a particular antibody to be therapeuticallyuseful to treat, liver fibrosis, for example, the antibody can be testedin vivo in a rat liver fibrosis model. Thus, preferred human antibodiesof the invention are able to block both human and rat TIMP-1 activity.If desired, human Fab TIMP-1 antibodies can be converted into fullimmunoglobulins, for example IgG₁ antibodies, before therapeuticassessment. This conversion is described in Example 5, below.

To identify antibodies that cross-react with human and rat TIMP-1, anELISA can be carried out using rat TIMP-1. Functional cross-reactivitycan be confirmed in an enzymatic assay, as described in Bickett et al.,Anal. Biochem. 212, 58-64, 1993. The assay uses human or rat TIMP-1,human MMP-1 or rat MMP-13 (the rat counterpart of human MMP-1), and asynthetic fluorogenic peptide substrate. Enzyme activity of uncomplexedMMP-1 (or MMP-13) is assessed by observing an increase in a fluorescencesignal.

Antibodies that block human and/or rat TIMP-1 activity can be screenedin an ELISA assay that detects the decrease of TIMP-1/MMP-1 complexformation in cultures of HepG2 cells. Antibodies that meet this criteriacan then be tested in a rat liver fibrosis model to assess therapeuticefficacy and correlate this efficacy with the ability of the antibodiesto block TIMP-1 inhibition of MMP-1 in vitro.

Antibodies that demonstrate therapeutic efficacy in the rat liverfibrosis model can then be tested for binding to and blockade of TIMP-2,-3, and -4 in an in vitro enzymatic assay. Blocking the minimum numberof TIMPs necessary for efficacy in liver fibrosis or otherTIMP-associated pathology is preferable to minimize potential sideeffects.

Polynucleotides Encoding Human TIMP-1 Antibodies

The invention also provides polynucleotides encoding human TIMP-1antibodies. These polynucleotides can be used, for example, to producequantities of the antibodies for therapeutic or diagnostic use.

Polynucleotides that can be used to encode the VHCDR3 regions shown inSEQ ID NOS:1-43 are shown in SEQ ID NOS:226-268, respectively.Polynucleotides that can be used to encode the VLCDR3 region shown inSEQ ID NOS:44-86 are shown in SEQ ID NOS:183-225, respectively.Polynucleotides that encode heavy chains (SEQ ID NOS:140-182) and lightchains (SEQ ID NOS:97-139) of human antibodies of the invention thathave been isolated from the MorphoSys HuCAL® library are shown in SEQ IDNOS:269-311 and SEQ ID NOS:312-354, respectively.

Polynucleotides of the invention present in a host cell can be isolatedfree of other cellular components such as membrane components, proteins,and lipids. Polynucleotides can be made by a cell and isolated usingstandard nucleic acid purification techniques, or synthesized using anamplification technique, such as the polymerase chain reaction (PCR), orby using an automatic synthesizer. Methods for isolating polynucleotidesare routine and are known in the art. Any such technique for obtaining apolynucleotide can be used to obtain isolated polynucleotides encodingantibodies of the invention. For example, restriction enzymes and probescan be used to isolate polynucleotides which encode the antibodies.Isolated polynucleotides are in preparations that are free or at least70, 80, or 90% free of other molecules.

Human antibody-encoding DNA molecules of the invention can be made withstandard molecular biology techniques, using mRNA as a template.Thereafter, DNA molecules can be replicated using molecular biologytechniques known in the art and disclosed in manuals such as Sambrook etal. (1989). An amplification technique, such as PCR, can be used toobtain additional copies of the polynucleotides.

Alternatively, synthetic chemistry techniques can be used to synthesizepolynucleotides encoding antibodies of the invention. The degeneracy ofthe genetic code allows alternate nucleotide sequences to be synthesizedthat will encode an antibody having, for example, one of the VHCDR3,VLCDR3, light chain, or heavy chain amino acid sequences shown in SEQ IDNOS:1-43, 44-86, 97-139, or 140-182, respectively.

Expression of Polynucleotides

To express a polynucleotide encoding a human antibody of the invention,the polynucleotide can be inserted into an expression vector thatcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods that are well known to thoseskilled in the art can be used to construct expression vectorscontaining sequences encoding human antibodies and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described, forexample, in Sambrook et al. (1989) and in Ausubel et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1995.See also Examples 1-3, below.

A variety of expression vector/host systems can be utilized to containand express sequences encoding a human antibody of the invention. Theseinclude, but are not limited to, microorganisms, such as bacteriatransformed with recombinant bacteriophage, plasmid, or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors,insect cell systems infected with virus expression vectors (e.g.,baculovirus), plant cell systems transformed with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids),or animal cell systems.

The control elements or regulatory sequences are those non-translatedregions of the vector—enhancers, promoters, 5′ and 3′ untranslatedregions—which interact with host cellular proteins to carry outtranscription and translation. Such elements can vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, can be used. For example, whencloning in bacterial systems, inducible promoters such as the hybridlacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.)or pSPORT1 plasmid (Life Technologies) and the like can be used. Thebaculovirus polyhedrin promoter can be used in insect cells. Promotersor enhancers derived from the genomes of plant cells (e.g., heat shock,RUBISCO, and storage protein genes) or from plant viruses (e.g., viralpromoters or leader sequences) can be cloned into the vector. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses are preferable. If it is necessary to generate a cell line thatcontains multiple copies of a nucleotide sequence encoding a humanantibody, vectors based on SV40 or EBV can be used with an appropriateselectable marker.

Large scale production of human TIMP-1 antibodies can be carried outusing methods such as those described in Wurm et al., Ann. N.Y. Acad.Sci. 782, 70-78, 1996, and Kim et al., Biotechnol. Bioengineer. 58,73-84, 1998.

Pharmaceutical Compositions

Any of the human TIMP-1 antibodies described above can be provided in apharmaceutical composition comprising a pharmaceutically acceptablecarrier. The pharmaceutically acceptable carrier preferably isnon-pyrogenic. The compositions can be administered alone or incombination with at least one other agent, such as stabilizing compound,which can be administered in any sterile, biocompatible pharmaceuticalcarrier, including, but not limited to, saline, buffered saline,dextrose, and water. A variety of aqueous carriers may be employed,e.g., 0.4% saline, 0.3% glycine, and the like. These solutions aresterile and generally free of particulate matter. These solutions may besterilized by conventional, well known sterilization techniques (e.g.,filtration). The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, etc. The concentration of theantibody of the invention in such pharmaceutical formulation can varywidely, i.e., from less than about 0.5%, usually at or at least about 1%to as much as 15 or 20% by weight and will be selected primarily basedon fluid volumes, viscosities, etc., according to the particular mode ofadministration selected. See U.S. Pat. No. 5,851,525. If desired, morethan one type of human antibody, for example with different K_(d) forTIMP-1 binding or with different IC₅₀s for MMP-inhibiting activityneutralization, can be included in a pharmaceutical composition.

The compositions can be administered to a patient alone, or incombination with other agents, drugs or hormones. In addition to theactive ingredients, these pharmaceutical compositions can containsuitable pharmaceutically-acceptable carriers comprising excipients andauxiliaries that facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Pharmaceuticalcompositions of the invention can be administered by any number ofroutes including, but not limited to, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, parenteral,topical, sublingual, or rectal means.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. Such labeling would include amount, frequency, and method ofadministration.

Methods of Decreasing MMP-Inhibiting Activity of Human TIMP-1

The invention provides methods of decreasing an MMP-inhibiting activityof human or rat TIMP-1. Such methods can be used therapeutically, asdescribed below, or in a research setting. Thus, the methods can becarried out in a cell-free system, in a cell culture system, or in vivo.In vivo methods of decreasing MMP-inhibiting activity of human or ratTIMP-1 are described below.

Human TIMP-1 is contacted with a human antibody that binds to the humanTIMP-1, thereby decreasing the MMP-inhibiting activity of the humanTIMP-1 relative to human TIMP-1 activity in the absence of the antibody.The antibody can be added directly to the cell-free system, cell culturesystem, or to an animal subject or patient, or can be provided by meansof an expression vector encoding the antibody.

Diagnostic Methods

The invention also provides diagnostic methods, with which human or ratTIMP-1 can be detected in a test preparation, including withoutlimitation a sample of serum, lung, liver, heart, kidney, colon, a cellculture system, or a cell-free system (e.g., a tissue homogenate). Suchdiagnostic methods can be used, for example, to diagnose disorders inwhich TIMP-1 is elevated. Such disorders include, but are not limitedto, liver fibrosis, alcoholic liver disease, cardiac fibrosis, acutecardiac syndrome, lupus nephritis, glomerulosclerotic renal disease,benign prostate hypertrophy, lung cancer, colon cancer, and idiopathicpulmonary fibrosis. When used for diagnosis, detection of an amount ofthe antibody-TIMP-1 complex in a test sample from a patient which isgreater than an amount of the complex in a normal sample identifies thepatient as likely to have the disorder.

The test preparation is contacted with a human antibody of theinvention, and the test preparation is then assayed for the presence ofan antibody-TIMP-1 complex. If desired, the human antibody can comprisea detectable label, such as a fluorescent, radioisotopic,chemiluminescent, or enzymatic label, such as horseradish peroxidase,alkaline phosphatase, or luciferase.

Optionally, the antibody can be bound to a solid support, which canaccommodate automation of the assay. Suitable solid supports include,but are not limited to, glass or plastic slides, tissue culture plates,microtiter wells, tubes, silicon chips, or particles such as beads(including, but not limited to, latex, polystyrene, or glass beads). Anymethod known in the art can be used to attach the antibody to the solidsupport, including use of covalent and non-covalent linkages, passiveabsorption, or pairs of binding moieties attached to the antibody andthe solid support. Binding of TIMP-1 and the antibody can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicrocentrifuge tubes.

Therapeutic Methods

The invention also provides methods of ameliorating symptoms of adisorder in which TIMP-1 is elevated. These disorders include, withoutlimitation, liver fibrosis alcoholic liver disease, cardiac fibrosis,acute coronary syndrome, lupus nephritis, glomerulosclerotic renaldisease, idiopathic pulmonary fibrosis, benign prostate hypertrophy,lung cancer, colon cancer, and scarring. See, e.g., Inokubo et al., Am.Heart J. 141, 211-17, 2001; Ylisirnio et al., Anticancer Res. 20,1311-16, 2000; Holten-Andersen et al., Clin. Cancer Res. 6, 4292-99,2000; Holten-Andersen et al., Br. J. Cancer 80, 495-503, 1999; Petersonet al., Cardiovascular Res. 46, 307-15, 2000; Arthur et al., Alcoholism:Clinical and Experimental Res. 23, 840-43, 1999; Iredale et al.,Hepatol. 24, 176-84, 1996.

Human antibodies of the invention are particularly useful for treatingliver fibrosis. All chronic liver diseases cause the development offibrosis in the liver. Fibrosis is a programmed uniform wound healingresponse. Toxic damage or injury caused by foreign proteins cause thedeposition of extracellular matrix such as collagen, fibronectin, andlaminin. Liver fibrosis and cirrhosis can be caused by chronicdegenerative diseases of the liver such as viral hepatitis, alcoholhepatitis, autoimmune hepatitis, primary biliary cirrhosis, cysticfibrosis, hemochromatosis, Wilson's disease, and non-alcoholicsteato-hepatitis, as well as chemical damage.

Altered degradation and synthesis of extracellular matrix (particularlycollagens) play central roles in pathogenesis of liver fibrosis. In theearly phases, hepatic stellate cells (HSC) are initially activated andrelease matrix metalloproteases with the ability to degrade the normalliver matrix. When HSC are fully activated, there is a netdown-regulation of matrix degradation mediated by increased synthesisand extracellular release of tissue inhibitors of metalloprotease(TIMP)-1 and -2. The dynamic regulation of activity of metalloproteasesduring liver fibrosis makes them and their inhibitors targets fortherapeutic intervention.

Human antibodies of the invention are also particularly useful fortreating lung fibrosis. Lung airway fibrosis is a hallmark of airwayremodeling in patients with chronic asthma, so human antibodies of theinvention are also particularly useful for chronic asthma. Airwayremodeling is a well-recognized feature in patients with chronic asthma.TIMP-1 but not TIMP-2 levels were significantly higher in untreatedasthmatic subjects than in glucocorticoid-treated subjects or controls(p<0.0001), and were far greater than those of MMP-1, MMP-2, MMP-3, andMMP-9 combined (Mautino et al., Am J Respir Crit. Care Med 1999160:324-330). TIMP-1 mRNA and protein expression are selectively andmarkedly increased in a murine model of bleomycin-induced pulmonaryfibrosis (Am. J. Respir. Cell Mol. Biol. 24:599-607, 2001). Thisspecific elevation of TIMP-1 without increase in MMPs in asthma patientssuggests that inhibition of TIMP-1 by an antibody can restore normalcollagen degradation in the lung.

Human antibodies of the invention are also particularly useful fortreating cancer. TIMP-1 protein has been found to be elevated in plasmaof colon (Holten-Andersen et al., Br J Cancer 1999, 80:495-503) andprostate (Jung et al., Int J Cancer, 1997, 74:220-223) cancer patients,and high TIMP-1 plasma level correlates with poor clinical outcome ofcolon cancer (Holten-Andersen et al., Clin Cancer Res 2000 6:4292-4299).TIMP-1 induces dose-dependent proliferation of breast tumorigenic clonalcell line and tyrosine phosphorylation (Luparello et al, Breast CancerRes Treat, 1999, 54:235-244). Therefore, the use of antibody againstTIMP-1 may block its ability to induce cancer.

Human TIMP-1 antibodies can be used to prevent or diminish scarformation, such as scar formation after surgery (particularly ophthalmicsurgery) or injury (such as a burn, scrape, crush, cut or tear injury).

In one embodiment of the invention, a therapeutically effective dose ofa human antibody of the invention is administered to a patient having adisorder in which TIMP-1 is elevated, such as those disorders describedabove. Symptoms of the disorder, including deposition of extracellularmatrix, as well as loss of tissue or organ function, are therebyameliorated.

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within thecapability of those skilled in the art. A therapeutically effective doserefers to that amount of human antibody that reduces MMP-inhibitingactivity of the TIMP-1 relative to the activity which occurs in theabsence of the therapeutically effective dose.

The therapeutically effective dose can be estimated initially either incell culture assays or in animal models, usually rats, mice, rabbits,dogs, or pigs. The animal model also can be used to determine theappropriate concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. A rat liver fibrosis model is described inExample 6.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeuticallyeffective in 50% of the population) and LD₅₀ (the dose lethal to 50% ofthe population) of a human antibody, can be determined by standardpharmaceutical procedures in cell cultures or experimental animals. Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD₅₀/ED₅₀.

Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the patient who requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the humanantibody or to maintain the desired effect. Factors that can be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions can be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

Polynucleotides encoding human antibodies of the invention can beconstructed and introduced into a cell either ex vivo or in vivo usingwell-established techniques including, but not limited to,transferrin-polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated cellular fusion,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, “gene gun,” and DEAE- orcalcium phosphate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 mgto about 50 mg/kg, about 50 mg to about 5 mg/kg, about 100 mg to about500 mg/kg of patient body weight, and about 200 to about 250 mg/kg ofpatient body weight. For administration of polynucleotides encoding theantibodies, effective in vivo dosages are in the range of about 100 ngto about 200 ng, 500 ng to about 50 mg, about 1 mg to about 2 mg, about5 mg to about 500 mg, and about 20 mg to about 100 mg of DNA.

The mode of administration of human antibody-containing pharmaceuticalcompositions of the invention can be any suitable route which deliversthe antibody to the host. Pharmaceutical compositions of the inventionare particularly useful for parenteral administration, i.e.,subcutaneous, intramuscular, intravenous, or intranasal administration.

All patents, patent applications, and references cited in thisdisclosure are expressly incorporated herein by reference. The abovedisclosure generally describes the present invention. A more completeunderstanding can be obtained by reference to the following specificexamples, which are provided for purposes of illustration only and arenot intended to limit the scope of the invention.

Example 1 Construction of a Human Combinatorial Antibody Library (HuCAL®Fab 1)

Cloning of HuCAL® Fab 1.

HuCAL® Fab 1 is a fully synthetic, modular human antibody library in theFab antibody fragment format. HuCAL® Fab 1 was assembled starting froman antibody library in the single-chain format (HuCAL®-scFv; Knappik etal., J. Mol. Biol. 296, 55, 2000). HuCAL® Fab 1 was cloned into aphagemid expression vector pMORPH® 18 Fab1 (FIG. 3). This vectorcomprises the Fd fragment with a phoA signal sequence fused at theC-terminus to a truncated gene III protein of filamentous phage, andfurther comprises the light chain VL-CL with an ompA signal sequence.Both chains are under the control of the lac operon. The constantdomains Cλ, Cκ, and CH are synthetic genes fully compatible with themodular system of HuCAL® (Knappik et al., 2000).

First, the Vλ and Vκ libraries were isolated from HuCAL®-scFv. Vλ1fragments were amplified by 15 PCR cycles (Pwo polymerase) with primers5′-GTGGTGGTTCCGATATC-3′ (SEQ ID NO:380) and5′-AGCGTCACA-CTCGGTGCGGCTTTCGGCTGGCCAAGAACGGTTA-3′ (SEQ ID NO:381).PCR-products were digested with EcoRV/DraIII and gel-purified.VLκ-chains were obtained by restriction digest with EcoRV/BsiWI andgel-purified. These Vλ and Vκ libraries were cloned into pMORPH® 18 Fab1cut with EcoRV/DraIII and EcoRV/BsiWI, respectively. After ligation andtransformation in E. coli TG-1, library sizes of 4.14×10⁸ and 1.6×10⁸,respectively, were obtained, in both cases exceeding the Vλ diversity ofHuCAL®-scFv.

Similarly, the VH library was isolated from HuCAL®-scFv by restrictiondigest using StyI/MunI. This VH library was cloned into the pMORPH®18-Vλ and Vκ libraries cut with StyI/MunI. After ligation andtransformation in E. coli TG-1, a total library size of 2.09×10¹⁰ wasobtained, with 67% correct clones (as identified by sequencing of 207clones).

Phagemid Rescue, Phage Amplification and Purification.

HuCAL® Fab was amplified in 2×TY medium containing 34 μg/mlchloramphenicol and 1% glucose (2×TY-CG). After helper phage infection(VCSM13) at 37° C. at an OD₆₀₀ of about 0.5, centrifugation andresuspension in 2×TY/34 μg/ml chloramphenicol/50 μg/ml kanamycin, cellswere grown overnight at 30° C. Phage were PEG-precipitated from thesupernatant (Ausubel et al., 1998), resuspended in PBS/20% glycerol, andstored at −80° C. Phage amplification between two panning rounds wasconducted as follows: mid-log phase TG1-cells were infected with elutedphage and plated onto LB-agar supplemented with 1% of glucose and 34μg/ml of chloramphenicol. After overnight incubation at 30° C., colonieswere scraped off and adjusted to an OD₆₀₀ of 0.5. Helper phage wereadded as described above.

Example 2 Solid Phase Panning

Wells of MaxiSorp™ microtiter plates (Nunc) were coated with rat- orhuman TIMP protein diluted to 50 μg/ml dissolved in PBS (2 μg/well).After blocking with 5% non-fat dried milk in PBS, 1-5×10¹² HuCAL® Fabphage purified as above were added for 1 h at 20° C. After severalwashing steps, bound phage were eluted by pH-elution with 100 mMtriethylamine and subsequent neutralization with 1M TRIS-Cl pH 7.0. SeeKrebs et al., J. Immunol. Meth. 254, 67, 2001. Two to three rounds ofpanning were performed with phage amplification conducted between eachround as described above.

Example 3 Solution Panning

Biotinylated antigen was diluted to 40 nM in PBS, 1013 HuCAL®-Fab 1phage were added and incubated for 1 h at 20° C. Phage-antigen complexeswere captured on Neutravidin plates (Pierce). After several washingsteps, bound phages were eluted by different methods (Krebs et al.,2001). Two rounds of panning were routinely performed.

Example 4 Subcloning of Selected Fab Fragments for Expression

The Fab-encoding inserts of the selected HuCAL® Fab 1 fragments weresubcloned into the expression vector pMORPH® x7_FS (Knappik et al., J.Mol. Biol. 296, 55, 2000) to facilitate rapid expression of soluble Fab.The DNA preparation of the selected HuCAL® Fab 1 clones was digestedwith XbaI/EcoRI, thus cutting out the Fab encoding insert (ompA-VL andphoA-Fd). Subcloning of the purified inserts into the XbaI/EcoRI cutvector pMORPH® x7, previously carrying a scFv insert, produces a Fabexpression vector designated pMORPH® x9_Fab1_FS (FIG. 4). Fabs expressedin this vector carry two C-terminal tags (FLAG™ and Strep-tagII) fordetection and purification.

Example 5 Identification of TIMP-Binding Fab Fragments by ELISA

The wells of 384-well Maxisorp ELISA plates were coated with 20 μl/wellsolutions of rat TIMP or human TIMP at a concentration of 5 μg/mldiluted in coating buffer. Expression of individual Fab in E. coli TG-1from expression vector pMORPH® x9_FS was induced with 0.5 mM IPTG for 12h at 30° C. Soluble Fab was extracted from the periplasm by osmoticshock (Ausubel et al., 1998) and used in an ELISA. The Fab fragment wasdetected after incubation with alkaline phosphatase-conjugated anti-Fabantibody (Dianova), followed by development with Attophos substrate(Roche) and measurement at Ex450 nm/Em535 nm. Values at 370 nm were readout after addition of horseradish peroxidase-conjugated anti-mouse IgGantibody and POD soluble substrate (Roche Diagnostics).

Example 6 Expression and Purification of HuCAL®-Fab 1 Antibodies in E.Coli

Expression of Fab fragments encoded by pMORPH® x9_FS in TG-1 cells wascarried out in shaker flask cultures with 1 liter of 2×TY mediumsupplemented with 34 μg/ml chloramphenicol. After induction with 0.5 mMIPTG, cells were grown at 22° C. for 16 h. Periplasmic extracts of cellpellets were prepared, and Fab fragments were isolated by Strep-tactin®chromatography (IBA, Goettingen, Germany). The apparent molecularweights were determined by size exclusion chromatography (SEC) withcalibration standards. Concentrations were determined byUV-spectrophotometry.

Example 7 Construction of HuCAL® Immunoglobulin Expression Vectors

Heavy chain cloning. The multiple cloning site of pcDNA3.1+ (Invitrogen)was removed (NheI/ApaI), and a stuffer compatible with the restrictionsites used for HuCAL® design was inserted for the ligation of the leadersequences (NheI/EcoRI), VH-domains (EcoRI/BlpI), and the immunoglobulinconstant regions (BlpI/ApaI). The leader sequence (EMBL M83133) wasequipped with a Kozak sequence (Kozak, 1987). The constant regions ofhuman IgG₁ (PIR J00228), IgG₄ (EMBL K01316), and serum IgA₁ (EMBLJ00220) were dissected into overlapping oligonucleotides with lengths ofabout 70 bases. Silent mutations were introduced to remove restrictionsites non-compatible with the HuCAL® design. The oligonucleotides werespliced by overlap extension-PCR.

Light Chain Cloning.

The multiple cloning site of pcDNA3.1/Zeo+ (Invitrogen) was replaced bytwo different stuffers. The κ-stuffer provided restriction sites forinsertion of a κ-leader (NheI/EcoRV), HuCAL®-scFv Vκ-domains(EcoRV/BsiWI) and the κ-chain constant region (BsiWI/ApaI). Thecorresponding restriction sites in the λ-stuffer were NheI/EcoRV(λ-leader), EcoRV/HpaI (Vλ-domains), and HpaI/ApaI (λ-chain constantregion). The κ-leader (EMBL Z00022) as well as the λ-leader (EMBLL27692) were both equipped with Kozak sequences. The constant regions ofthe human κ- (EMBL J00241) and λ-chain (EMBL M18645) were assembled byoverlap extension-PCR as described above.

Generation of IgG-Expressing CHO-Cells.

CHO-K1 cells were co-transfected with an equimolar mixture of IgG heavyand light chain expression vectors. Double-resistant transfectants wereselected with 600 μg/ml G418 and 300 μg/ml Zeocin (Invitrogen) followedby limiting dilution. The supernatant of single clones was assessed forIgG expression by capture-ELISA (see below). Positive clones wereexpanded in RPMI-1640 medium supplemented with 10% ultra-low IgG-FCS(Life Technologies). After adjusting the pH of the supernatant to 8.0and sterile filtration, the solution was subjected to standard protein Acolumn chromatography (Poros 20 A, PE Biosystems).

Example 8 Design of the CDR3 Libraries

Vλ Positions 1 and 2.

The original HuCAL® master genes were constructed with their authenticN-termini: Vλ11: QS (CAGAGC), Vλ12: QS (CAGAGC), and Vλ13: SY (AGCTAT).Sequences containing these amino acids are shown in WO 97/08320. DuringHuCAL® library construction, the first two amino acids were changed toDI to facilitate library cloning (EcORI site). All HuCAL® librariescontain Vλ1 genes with the EcoRV site GATATC (DI) at the 5′-end. AllHuCAL® kappa genes (master genes and all genes in the library) containDI at the 5′-end.

VH Position 1.

The original HuCAL® master genes were constructed with their authenticN-termini: VH1A, VH1B, VH2, VH4, and VH6 with Q (=CAG) as the firstamino acid and VH3 and VH5 with E (=GAA) as the first amino acid.Sequences containing these amino acids are shown in WO 97/08320. In theHuCAL® Fab 1 library, all VH chains contain Q (=CAG) at the firstposition.

Vκ1/Vκ3 Position 85.

Because of the cassette mutagenesis procedure used to introduce the CDR3library (Knappik et al., J. Mol. Biol. 296, 57-86, 2000), position 85 ofVκ1 and Vκ3 can be either T or V. Thus, during HuCAL® scFv 1 libraryconstruction, position 85 of Vκ1 and Vκ3 was varied as follows: Vκ1original, 85T (codon ACC); Vκ1 library, 85T or 85V (TRIM codons ACT orGTT); Vκ3 original, 85V (codon GTG); Vκ3 library, 85T or 85V (TRIMcodons ACT or GTT); the same applies to HuCAL® Fab1.

CDR3 Design.

All CDR3 residues which were kept constant are indicated in FIG. 1.

CDR3 Length.

The designed CDR3 length distribution is as follows. Residues which werevaried are shown in brackets (x) in FIG. 1. V kappa CDR3, 8 amino acidresidues (position 89 to 96) (occasionally 7 residues), with Q90 fixed;V lambda CDR3, 8 to 10 amino acid residues (position 89 to 96)(occasionally 7-10 residues), with Q89, S90, and D92 fixed; and VH CDR3,5 to 28 amino acid residues (position 95 to 102) (occasionally 4-28),with D101 fixed.

Example 9 Chronic Carbon Tetrachloride-Induced Liver Fibrosis

Sprague Dawley rats (200-220 g) are used in an in vivo model of liverfibrosis. To maximally induce microsomal metabolism of carbontetrachloride metabolism, animals receive 1 g/l isoniazid with theirdrinking water starting one week before the administration of carbontetrachloride. Carbon tetrachloride (1:1 in mineral oil) is administeredorally every fifth day at a dose of 0.2 ml/100 g body weight. A humanTIMP-1 antibody is administered intravenously, either once orrepeatedly, during the period of carbon tetrachloride treatment.Necropsy is performed after 5-7 weeks of treatment. McLean et al., Br.J. Exp. Pathol. 50, 502-06, 1969.

Transverse cylinders of liver tissue are cut from the right liver lobe,fixed in formaldehyde, and embedded in paraffin. The amount of fibrosisin the liver is indicated by the picrosirius red-stained fibrotic areas.Picrosirius-positive areas are determined in several centrilobularfields in each section. Parameters of color detection are standardizedand kept constant throughout the experiment. The field are selectedusing a standardized grid which covers an area of 31 mm2. A LeicaQuantimed 500 MC system is used for morphometry.

Example 10 Hydroxyproline Determination

The method of Prockop & Udenfried, Anal. Biochem. 1, 228-39, 1960, canbe used to determine hydroxyproline is liver tissues, with the followingmodifications. Liver specimens of 60-90 mg wet weight are dried andhydrolyzed in 6 N HCl at 100° C. for 17 h. The hydrolyzed material isdried and reconstituted in 5 ml of deionized water. Two hundredmicroliters of this hydrolysate are mixed with 200 ml of ethanol and 200ml chloramin T solution (0.7% in citrate buffer [5.7 g sodium acetate,3.75 g trisodium citrate, 0.55 g citric acid, 38.5 ml ethanol, made upto 100 ml with water]) and allowed to oxidize for 20 min at roomtemperature. Four hundred microliters of Ehrlich's reagent (12 gp-dimethylaminobenzldehyde in 40 ml ethanol and 2.7 ml H₂SO₄) are added.After incubation for 3 h at 35° C., absorbance at 573 nm is measured.

Example 11 Affinity Determination by Surface Plasmon ResonanceMeasurements (Biacore™)

For affinity determination, monomeric fractions of affinity and SECpurified Fab fragments or purified IgG1 molecules were used. Allexperiments were conducted in HBS buffer at a flow rate of 20 μl/min at25° C. on a BIAcore™ instrument. Antigens in 100 mM sodium acetate pH5.0 were coupled to a CM 5 sensor chip using standard EDC-NHS couplingchemistry. Applying 3-4 μl of 5 μg/ml TIMP-1 typically resulted in 500resonance units for kinetic measurements. All sensograms were fittedglobally using BIA evaluation software. For monovalent Fab fragments amonovalent fit (Langmuir binding) and for IgGs a bivalent fit wasapplied.

Example 12 IC₅₀ Determination in Human TIMP-1/Human MMP-1 and RatTIMP-1/Rat MMP-13 Assay

Purified Fab fragments or IgGs were used for IC₅₀ determination.Antibodies were diluted in triplicate to the indicated concentrations inassay buffer containing 0.05% BSA. After addition of TIMP (final conc.1.2 nM or 0.4 nM for modified in human TIMP-1/human MMP-1 assay), MMP(final conc. 1.2 nM or 0.4 nM for modified in human TIMP-1/human MMP-1assay), and peptide substrate (final conc. 50 μM) and incubation for 1-3h at 37° C., fluorescence at Ex320 nm/Em430 nm was measured.

The following controls were included in the assay and used as referencevalues for IC₅₀ determination:

-   A: MMP+substrate: this value was defined as 100% MMP activity in    absence of antibody and TIMP.-   B: MMP+TIMP+substrate: this value was defined as maximum inhibition    achieved in the assay and calculated as a % of total MMP activity.

To define the concentration of antibody that resulted in 50% reversal ofinhibition (IC₅₀), the following procedure was used:

-   -   The value for 50% reversal of inhibition (expressed as %        activity MMP) was calculated as: Y=[(A−B)/2]+B.    -   MMP activity was plotted against concentration of antibody in        the assay.    -   The concentration of antibody that results in 50% reversal of        inhibition (Y) was read on the x-axis and defined as IC₅₀.    -   Error bars in the graphs were derived from triplicate wells in        one assay.    -   Standard deviations for IC₅₀ values were calculated from 3        independent assays.

Example 13 Affinity Maturation of Selected Fab by Stepwise Exchange ofCDR Cassettes

To increase affinity and biological activity of selected antibodyfragments, CDR regions were optimized by cassette mutagenesis usingtrinucleotide directed mutagenesis (Virnekäs et al., 1994). Fabfragments in expression vector pMORPH® x9 were cloned into phagemidvector pMORPH®_(—)18 using EcoRI/XbaI restriction sites. CDR cassettescontaining several diversified positions were synthesized and clonedinto Fab fragments in pMORPH®_(—)18 using unique restriction sites(Knappik et al., 2000). Affinity maturation libraries were generated bytransformation into E. coli TOP10F, and phage were prepared as describedabove. Phage displaying Fab fragments with improved affinity wereselected by 2-3 rounds solution panning using stringent washingconditions (e.g., competition with 1 μM non-biotinylated antigen orwashing for up to 48 h with frequent buffer exchange) and limitedamounts of antigen (0.04-4 nM). Seventeen human TIMP-1 antibodies weretested for affinity to human TIMP-1 (with some tested for affinity torat TIMP-1) using a BIAcore™ assay. The K_(d) of these antibodies forhuman TIMP-1 and rat TIMP-1 are shown in Table 1.

TABLE 1 Overview of species cross-reactive Fab Monovalent K_(D)Monovalent K_(D) IC₅₀ in human IC₅₀ in rat Fab human TIMP-1 rat TIMP-1protease assay protease assay MS-BW-25 25 +/− 16 nM* 4517 +/− 2400 nM115 +/− 15 nM >300 nM MS-BW-27 ~74 nM ~3200 nM Non blocking MS-BW-21 520+/− 20 nM 36 +/− 2 nM >300 nM 67 +/− 5 nM MS-BW-38 ~3 nM ~353 nM ~11nM >300 nM MS-BW-39 ~7500 nM ~108 nM >100 nM >100 nM *In cases werestandard deviations are given, three independent measurements were donewith Fab from three different protein expressions/purifications.~Indicates preliminary data, in cases where measurement was done onlyonce.

Example 14 Screening for Fab with Improved Off-Rates by Koff RankingUsing Surface Plasmon Resonance

Phage eluted after solution panning were used to infect E. coli TG-1 andplated on agar plates containing 34 μg/ml chloramphenicol. Clones werepicked into 96 well plates and used to produce Fab fragments. On thesame plate, parental clones were inoculated as controls. Soluble Fab wasextracted from the periplasm by osmotic shock (Ausubel et al., 1998) andused for koff ranking in BIAcore™

All measurements were conducted in HBS buffer at a flow rate of 20μl/min at 25° C. on a BIAcore™ instrument. Antigens in 100 mM sodiumacetate pH 4.5 were coupled to a CM 5 sensor chip using standard EDC-NHScoupling chemistry. Applying 10 μl of 25 μg/ml TIMP-1 typically resultedin 5000 resonance units for koff ranking All sensograms were fittedusing BIA evaluation software. Clones with improved off rate wereselected by comparison to parental clones.

Example 15 Generation of Species Cross-Reactive Antibodies

To maximize the likelihood of obtaining blocking antibodies that arecross-reactive between human and rat TIMP-1, alternating pannings werecarried out on rat and human protein. Additionally, all antibodiesselected by pannings on solely the human or rat TIMP-1 protein wereanalyzed for cross-reactivity in order to check for cross-reactiveantibodies that might be selected by chance. Antibodies selected fromthese pannings were analyzed for cross-reactivity in ELISA using crudeE. coli extracts. Cross-reactive antibodies in this assay were subjectedto expression in 1-liter scale followed by purification. Purifiedantibodies were tested for cross-reactivity in BIAcore™ and proteaseassays (Table 1).

As shown in Table 1, a total of five different Fab cross-reactive withhuman and rat TIMP-1 were generated. BIAcore™ measurements revealed thatalthough these antibodies clearly bind to human and rat TIMP-1,affinities for both species differ by at least a factor of 50. Anantibody used for human therapy or in an animal model should have anaffinity to the target protein in the low nanomolar, preferably in thesub-nanomolar range. As none of the above-described antibodies hadaffinities in this range for both species, these antibodies were notconsidered useful for further experiments or development.

Example 16 Generation of Blocking Antibodies Against Human TIMP-1

To generate blocking antibodies against human TIMP-1, the HuCAL®-Fab 1library was used for antibody selection (AutoPan®) on purified TIMP-1protein followed by subcloning and expression of the selected Fabfragments in E. coli. Crude antibody-containing E. coli extracts wereused for primary antibody characterization in ELISA (AutoScreen®).Purified Fab proteins were subjected to further characterization inELISA, TIMP-1/MMP-1 assay and BIAcore™. A total of 6100 clones wereanalyzed in AutoScreen®, 670 of them showed binding to human TIMP-1.Sequence analysis revealed that in total seven unique antibody cloneshad been selected (Table 2). For these seven Fab clones, the affinitiesmeasured in BIAcore™ were in the range of 10-180 nM (Table 4). Whentested in the human protease assay, five of them were able to block theinteraction between human TIMP-1 and MMP-1. The concentration ofmonovalent Fab needed to reverse the inhibitory effect of human TIMP-1on human MMP-1 activity by 50% (IC₅₀) was in the range of 11-100 nM(Table 2). The most active Fab clones are MS-BW-3 (K_(d) 13 nM; IC₅₀ 11nM) and MS-BW-28 (K_(d) 10 nM; IC₅₀ 22 nM).

A striking feature of antibodies selected against human TIMP-1 is thatthey all exhibit the combination VH312 and a relatively short VH-CDR3region, predominantly four amino acids (see Table 2). The HCDR3cassettes assembled for the HuCAL®-Fab 1 library were designed toachieve a length distribution ranging from 5 to 28 amino acid residues.A four amino acid HCDR3 can occur in the library due to TRIM deletion,but is considered a very rare event. Another remarkable feature was thehigh degree of sequence homology among the selected LCDR3 sequences.

TABLE 2  Overview of anti-human TIMP-1 Fab Framework + CDR 3 sequenceMonovalent K_(D) IC₅₀ in human Fab VH HCDR3 VL LCDR3 to human TIMP-1protease assay MS-BW-1 H3 FMDI, λ2 QSYDYQQFT,  65 +/− 13 nM* >100 nMSEQ ID NO: 1 SEQ ID NO: 44 MS-BW-2 H3 GFDY, λ2 QSYDFKTYL, 180 +/− 28nM >100 nM SEQ ID NO: 2 SEQ ID NO: 45 MS-BW-3 H3 FLDI, λ2 QSYDFLRFS, 13 +/− 2 nM  11 +/− 2 nM SEQ ID NO: 3 SEQ ID NO: 46 MS-BW-25 H3TFPIDADS, λ2 QSYDFINVI,  25 +/− 16 nM 115 +/− 15 nM SEQ ID NO: 4SEQ ID NO: 47 MS-BW-26 H3 GHVDY, λ2 QSYDFVRFM,  ~100 nM non blockingSEQ ID NO: 5 SEQ ID NO: 48 MS-BW-27 H3 YWRGLSFDI, λ2 QSYDFYKFN,  ~74non blocking SEQ ID NO: 6 SEQ ID NO: 49 MS-BW-28 H3 FFDY, λ2 QSYDFRRFS, 10 +/− 1 nM  22 +/− 2 nM SEQ ID NO: 7 SEQ ID NO: 50 *In cases werestandard deviations are given, three independent measurements were donewith Fab from three different protein expressions/purifications.~Indicates preliminary data, in cases where measurement was done onlyonce.

Example 17 Increasing the Affinity of Selected Anti-Human TIMP-1Antibodies

In order to increase the affinity of monovalent anti-human TIMP-1 Fabfragments to the sub-nanomolar range, a step-wise affinity maturationapproach was applied, by optimizing CDR sequences and keeping frameworkregions constant.

Affinity Maturation by Light Chain Cloning

The CDR3 sequences of the two antibody fragments with highest affinity(MS-BW-3 and MS-BW-28) had the remarkable feature of an unusually shortfour amino acid HCDR3 sequence. Furthermore, each Fab had a very similarLCDR3 sequence. This indicates that MS-BW-3 and MS-BW-28 bind to thesame epitope and that this epitope might tolerate only a very smallsubset of CDR3 sequences. As a four amino acid HCDR3 is a very rareevent in the library, it can be anticipated that in the initial librarynot all possible combinations of the short HCDR3 and the preferred LCDR3are present. Therefore, it was considered that another combination ofthe selected HCDR3 and LCDR3 sequences might increase the affinity. Forthis approach, the heavy chain of MS-BW-3 and MS-BW-28 were paired withthe light chains of MS-BW-1, -2, -3, -25, -26, -27, and -28 by cloning.

The resulting constructs were transformed into E. coli andexpressions/purifications in 1-liter scale were performed. Of the 12 newconstructs, 10 resulted in functional Fab molecules. These were analyzedin BIAcore™ and human protease assay as summarized in Table 3. The bestantibody named MS-BW-44 had a monovalent affinity of 2 nM and an IC50 of4 nM (FIG. 7) and was thus improved by a factor of 6.5 (K_(d)) or 2.75(IC₅₀).

TABLE 3  Overview of Fab derived from light chain cloning Framework +CDR 3 sequence Monovalent K_(D) IC₅₀* in human Fab VH HCDR3 VL LCDR3to human TIMP-1 protease assay MS-BW-43 H3 FLDI, λ2 QSYDYQQFT,  ~49nM >100 nM SEQ ID NO: 3 SEQ ID NO: 44 MS-BW-44 H3 FLDI, λ2 QSYDFKTYL,  ~6 nM 29 +/− 6 nM SEQ ID NO: 3 SEQ ID NO: 45 MS-BW-45 H3 FLDI,  λ2QSYDFINVI,  ~65 nM >100 nM SEQ ID NO: 3 SEQ ID NO: 47 MS-BW-46 H3 FLDI,λ2 QSYDFVRFM, 2 +/− 0.4 nM*  4 +/− 1 nM SEQ ID NO: 3 SEQ ID NO: 48MS-BW-47 H3 FLDI, λ2 QSYDFYKFN, 8 +/− 5 nM  9 +/− 3 nM SEQ ID NO: 3SEQ ID NO: 49 MS-BW-48 H3 FLDI, λ2 QSYDFRRFS, 6 +/− 3 nM  4 +/− 0.5 nMSEQ ID NO: 3 SEQ ID NO: 50 MS-BW-49 H3 FFDY, λ2 QSYDYQQFT, ~152 nM >100nM SEQ ID NO: 7 SEQ ID NO: 44 MS-BW-50 H3 FFDY, λ2 QSYDFKTYL,  ~21nM >100 nM SEQ ID NO: 7 SEQ ID NO: 45 MS-BW-51 H3 FFDY, λ2 QSYDFINVI,  ~7 nM  7 +/− 1 nM SEQ ID NO: 7 SEQ ID NO: 47 MS-BW-52 H3 FFDY, λ2QSYDFVRFM,  ~11 nM  9 +/− 1 nM SEQ ID NO: 7 SEQ ID NO: 48 *In cases werestandard deviations are given, three independent measurements were donewith Fab from three different protein expressions/purifications.~Indicates preliminary data, in cases where measurement was done onlyonce.

Affinity Maturation by Optimizing HCDR1 and HCDR2

In the HuCAL®-Fab 1 library, only the CDRs HCDR3 and LCDR3 arediversified to a high extent. Although it is known from crystallographicstudies that amino acids from these two CDRs make most of the antibodyantigen contacts, the residual four CDRs are also important for antigenbinding. However, their contribution to the binding energy can vary fromantibody to antibody. In the HuCAL®-Fab 1 library those CDRs exhibitonly a limited variability due to the presence of the different masterframeworks (Knappik et al., 2000). In order to improve the affinity ofthe selected antibodies, an affinity maturation approach by randomizingHCDR1 and HCDR2 was applied. For this approach two affinity maturationlibraries based on MS-BW-44 cloned into phage display vector pMORPH® 18were created. In library 1, only HCDR2 of MS-BW-44 was diversified using“TRIM technology” as described in Virnekäs et al., Nucl. Acids. Res. 22,5600-07, 1994; Knappik et al., J. Mol. Biol. 296, 57-86, 2000. Inlibrary 2, both HCDR1 and HCDR2 were diversified using the TRIMtechnology. In both cases, phage antibody libraries comprising 1×10⁸different clones were obtained. Both libraries were mixed and used asinput for a modified AutoPan® procedure. In order to select antibodieshaving an increased affinity to human TIMP-1, solution panning usinglimiting amounts of biotinylated antigen and stringent washingconditions were applied. Antibody off rates were ranked by BIAcore™using crude E. coli extracts of selected antibodies. Clones with sloweroff rate than parental clone MS-BW-44 were subjected to 1-liter scaleexpression and purification. Purified Fab were analyzed in BIAcore™ andhuman protease assay (Table 4).

TABLE 4 Comparison of Fab derived from HCDR1 and HCDR2 optimization withparental clone MS-BW-44 Monovalent K_(D) IC₅₀ in human Fab to humanTIMP-1 protease assay* MS-BW-44  2 +/− 0.4 nM  2 +/− 0.5 nM MS-BW-44-20.5 +/− 0.2 nM 0.4 +/− 0.3 nM MS-BW-44-6 0.6 +/− 0.2 nM 0.2 +/− 0.1 nM*IC₅₀ values derived from modified protease assay using decreasedamounts of TIMP-1 and MMP-1 (0.4 nM each).

Clone MS-BW-44-2 was derived from library 1 thus having a modified HCDR2cassette. Its affinity measured by BIAcore™ was 0.5 nM. Clone MS-BW-44-6was derived from library 2 having a modified HCDR 1 and HCDR 2 cassetteand the affinity measured by BIAcore™ was 0.6 nM. A sequence comparisonbetween the affinity matured antibodies and their parental clones isshown in Table 8.

TABLE 8 Overview and sequence comparison of affinity matured Fab fragments against humanTIMP-1. Sequence changes compared to parental Fab fragments (bold) are italicizedVH Clone HCDR1 HCDR2 HCDR3 VL MS- Frame- sequence sequence sequenceFrame- BW- work (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) work  3 VH3GFTFSSYAMS AISGSGGSTYYADSVKG FLDI (3) VL2 (355) (357) 44 VH3 GFTFSSYAMSAISGSGGSTYYADSVKG FLDI (3) VL2 (355) (357) 44-6 VH3 GFTFNSYAMSVISGNGSNTYYADSVKG FLDI (3) VL2 (356) (358) 44-2 VH3 GFTFSSYAMSGISGNGVLIFYADSVKG FLDI (3) VL2 (355) (359) 44-2-4  VH3 GFTFSSYAMSGISGNGVLIFYADSVKG GLMDY (360) VL2 (355) (359) 44-2-15 VH3 GFTFSSYAMSGISGNGVLIFYADSVKG WFDH (361) VL2 (355) (359) 44-2-16 VH3 GFTFSSYAMSGISGNGVLIFYADSVKG WFDV (362) VL2 (355) (359) 44-6-1  VH3 GFTFNSYAMSVISGNGSNTYYADSVKG FLDI (3) VL2 (356) (358) VL Monov. K_(D) IC₅₀ in CloneLCDR1 LCDR2 LCDR3 to human human MS- sequence sequence sequence TIMP-1protease BW- (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (nM) assay (nM)  3TGTSSDVGGYNYVS DVSNRPS QSYDFLRFS 13 +/− 2  11 +/− 2    (363) (364) (47)44 TGTSSDVGGYNYVS DVSNRPS QSYDFVRFM   2 +/− 0.4 4 +/− 1   (363) (364)(48) 44-6 TGTSSDVGGYNYVS DVSNRPS QSYDFVRFM 0.6 +/− 0.2 0.2 +/− 0.1 *(363) (364) (48) 44-2 TGTSSDVGGYNYVS DVSNRPS QSYDFVRFM 0.5 +/− 0.20.4 +/− 0.3 * (363) (364) (48) 44-2-4  TGTSSDVGGYNYVS DVSNRPS QSYDFVRFM 0.2 +/− 0.02 0.2 +/− 0.1 * (363) (364) (48) 44-2-15 TGTSSDVGGYNYVSDVSNRPS QSYDFVRFM 0.3 +/− 0.1 0.2 +/− 0.1 * (363) (364) (48) 44-2-16TGTSSDVGGYNYVS DVSNRPS QSYDFVRFM 0.5 +/− 0.2 0.3 +/− 0.1 * (363) (364)(48) 44-6-1  TGTSSDVGGYNYVS DVSNRPS QSYDFIRFM  0.2 +/− 0.04 0.2 +/−0.1 * (363) (364) (365) * IC₅₀ values derived from modified proteaseassay using decreased amounts of TIMP-1 and MMP-1; IC₅₀ of MS-BW-44 is2nM under these conditions

When initially analyzed in the human TIMP-1/MMP-1 assay, it was notpossible to distinguish a Fab with a sub-nanomolar affinity from a Fabwith 1 nM affinity, most likely because the concentration of Fabrequired to reverse the inhibitory effect of human TIMP-1 on human MMP-1activity by 50% was below the concentration of total TIMP-1 in theassay. When a modified assay was used with concentrations of TIMP-1 andMMP-1 decreased from 1.2 nM to 0.4 nM, it was possible to distinguish a2 nM Fab from a sub-nanomolar Fab (Table 4, FIG. 8). Using this modifiedprotease assay, MS-BW-44-2 and MS-BW-44-6 had IC₅₀ values of 0.4 nM and0.2 nM respectively. Parental clone MS-BW-44 had an IC₅₀ of 2 nM underthese conditions. Thus, by this affinity maturation approach, anaffinity gain of a factor of 5 (K_(d)) or 5-10 (IC₅₀) was achieved.

Affinity Maturation by Optimizing HCDR3

As mentioned above, amino acid residues in HCDR3 and LCDR3 areconsidered the most important for antigen binding. Taking into accountthat a four amino acid HCDR3 was not planned in the design of HuCAL®-Fab1 and thus only occurs as a rare case due to a TRIM deletion, probablynot all possible combinations of the four amino acids in HCDR3 wererepresented in the original HuCAL®-Fab 1 library. Therefore, an affinitymaturation library was constructed with four and five amino acid HCDR3maturation cassettes inserted into Fab derived from the previousmaturation cycle (among them MS-BW-44-2 and MS-BW-44-6). The obtainedaffinity maturation library had a diversity of 1×10⁸ clones, thereforetheoretically covering all possible four and five amino acid HCDR3variations. Applying very stringent panning conditions, the bestantibody identified, MS-BW-44-2-4, had an affinity measured by BIAcore™of 0.2 nM and an IC₅₀ in human TIMP-1/MMP-1 assay of 0.2 nM. A sequencecomparison between the affinity matured antibodies and their parentalclones is shown in Table 8. The improvement factor gained by thisaffinity maturation approach is 2.5 with respect to the affinity and 2with respect to the IC₅₀.

Affinity Maturation by Optimizing LCDR3

As an alternative approach, a maturation strategy was used to furtheroptimize the light chain CDR3 sequence. This was due to the fact that inthe first maturation cycle where light chain exchange cloning betweenselected antibodies was applied, only a very limited subset of sequencevariation had been exploited. Therefore, a maturation library wasconstructed in which, using TRIM technology, a diversified LCDR3cassette was inserted into Fab derived from HCDR1 and HCDR2 optimization(among them MS-BW-44-2 and MS-BW-44-6). The best Fab identified withthis maturation strategy was MS-BW-44-6-1 with an affinity measured byBIAcore™ of 0.15 nM and an IC₅₀ in a human TIMP-1/MMP-1 assay of 0.2 nM.A sequence comparison between the affinity matured antibody and itsparental clones is shown in Table 8. The improvement factor gained bythis maturation approach is 4 with respect to affinity. A furtherimprovement of the IC₅₀ in the protease assay could not be measured dueto limitations in the assay.

As a result of a step-wise affinity maturation approach using fourdifferent maturation strategies, the monovalent affinity of ananti-human TIMP-1 specific Fab fragment was improved by a factor of 87and its activity in human TIMP-1/MMP-1 assay by a factor of 55. Thedecision for defining the best Fab fragment has been made on the basisof K_(d) measurements using BIAcore™, as this method proved to bereliable for ranking antibodies with sub-nanomolar affinities, whereasthe sensitivity of the human TIMP-1/MMP-1 assay was considered notsuitable to rank activity of the best Fabs in the sub-nanomolar rangewith respect to each other.

The best Fab MS-BW-44-6-1 has an affinity measured by BIAcore™ of 0.15nM and an IC₅₀ in human TIMP-1/MMP-1 assay of 0.2 nM. Compared to itsparental clone, MS-BW-3, it has optimized LCDR3, HCDR1 and HCDR2sequences.

Example 18 Cross Reactivity of Selected Anti-Human TIMP-1 Fab withTIMP-2, TIMP-3, and TIMP-4

TIMP-1 belongs to a family of closely related protease inhibitors allbinding to various members of the MMP family of proteases. To date thereare four human TIMP proteins described. To investigate potentialcross-reactivity of antibody fragments selected against human TIMP-1with other members of the human TIMP family, an ELISA was performed inwhich binding of antibody fragments to immobilized purified humanTIMP-1, -2, -3 or -4 was analyzed (FIG. 10). Antibody fragments bindingto immobilized human TIMP-1 showed no binding to human TIMP-2, -3, -4above background level when compared to unrelated control protein BSA.

Example 19 Generation of Blocking Antibodies Against Rat TIMP-1

To generate blocking antibodies against rat TIMP-1, the HuCAL®-Fab 1library was used for antibody selection (AutoPan®) on immobilized ratTIMP-1 followed by subcloning and expression of the selected Fabfragments in E. coli. Crude antibody-containing E. coli extracts wereused for primary antibody characterization in ELISA (AutoScreen®).Purified Fab proteins were subjected to further characterization inELISA, protease assays, and BIAcore™. Of the 8,450 selected clones wereanalyzed in AutoScreen®, 750 of them showed binding to rat TIMP-1.Sequence analysis revealed that in total 36 unique Fab clones specificfor rat TIMP-1 were enriched during selection (Table 7). Theiraffinities were measured by BIAcore™ and were found to be in the rangeof 9-1000 nM (Table 7). When tested in the rat protease assay, all butone of them were able to block the interaction between rat TIMP-1 andrat MMP-13 (Table 7). The concentration of monovalent Fab needed toreverse the inhibitory effect of rat TIMP-1 on rat MMP-13 activity by50% (IC₅₀) was in the range of 7-300 nM. The most active Fab clones areMS-BW-14 (K_(d) 10 nM; IC₅₀ 14 nM), MS-BW-17 (K_(d) 13 nM; IC₅₀ 11 nM),and MS-BW-54 (K_(d) 9 nM; IC₅₀ 7 nM).

TABLE 7  Overview of anti-rat TIMP-1 Fab Framework + CDR 3 sequenceMonovalent K_(D) IC₅₀* in rat Fab VH HCDR3 VL LCDR3 to rat TIMP-1protease assay MS-BW-5 H1A GLYWAVYPYFDF, λ1 QSRDFNRGP, ~210 nMnon blocking SEQ ID NO: 8 SEQ ID NO: 51 MS-BW-6 H3 LDTYYPDLFDY, λ1QSYDQRKW,  ~68 nM ~100 nM SEQ ID NO: 9 SEQ ID NO: 52 MS-BW-7 H1ATYYYFDS, κ3 QQLYGTVS,  ~168 nM >300 nM SEQ ID NO: 10 SEQ ID NO: 53MS-BW-9 H3 YMAYMAEAIDV, λ1 QSYDGFKTH, ~256 nM >300 nM SEQ ID NO: 11SEQ ID NO: 54 MS-BW-10 H1B LVGIVGYKPDELLYFDV, λ3 QSYDYSLL, ~200 nM  ~30nM SEQ ID NO: 12 SEQ ID NO: 55 MS-BW-11 H3 YGAYFGLDY, λ3 QSYDFNFH, ~200nM >300 nM SEQ ID NO: 13 SEQ ID NO: 56 MS-BW-12 H6 GYADISFDY, λ2QSYDMIARYP, ~419 nM >300 nM SEQ ID NO: 14 SEQ ID NO: 57 MS-BW-13 H3YYLLLDY, λ3 QSWDIHPFDV, ~939 nM not tested SEQ ID NO: 15 SEQ ID NO: 58MS-BW-14 H1A WSDQSYHYYWHPYFDV, λ1 QSWDLEPY,  10 +/− 5 nM 14 +/− 3 nMSEQ ID NO: 16 SEQ ID NO: 59 MS-BW-15 H3 LIGYFDL,  λ2 QSYDVLDSE,  ~80 nM~200 nM SEQ ID NO: 17 SEQ ID NO: 60 MS-BW-17 H5 LTNYFDSIYYDH, λ2QSYDPSHPSK,  13 +/− 3 nM 11 +/− 3 nM SEQ ID NO: 18 SEQ ID NO: 61MS-BW-18 H5 LVGGGYDLMFDS, λ2 QSYDDMQF, ~159 nM >300 nM SEQ ID NO: 19SEQ ID NO: 62 MS-BW-19 H5 YVTYGYDDYHFDY, λ2 QSWDINHAI, ~187 nM >300 nMSEQ ID NO: 20 SEQ ID NO: 63 MS-BW-20 H1A SGYLDY, λ2 QSYDYYDYG,  ~70nM >300 nM SEQ ID NO: 21 SEQ ID NO: 64 MS-BW-21 H1A YIGYTNVMDIRPGYFLDY,κ3 QQANDFPI,  36 +/− 2 nM  67 +/− 5 nM SEQ ID NO: 22 SEQ ID NO: 65MS-BW-22 H5 FRAYGDDFYFDV, λ2 QSWDNLKMPV, 35 nM 65 +/− 11 nMSEQ ID NO: 23 SEQ ID NO: 66 MS-BW-23 H1B JMWSDYGQLVKGGDI, λ2 QSYDVFPINR,~207 nM >300 nM SEQ ID NO: 24 SEQ ID NO: 67 MS-BW-24 H5 YYVTDTAYFDY, λ2QSDLYFP,   23 nM 20 +/− 1 nM SEQ ID NO: 25 SEQ ID NO: 68 MS-BW-29 H5HDFDGSIFMDF, λ2 QSYDVTPR, ~214 nM >100 nM SEQ ID NO: 26 SEQ ID NO: 69MS-BW-30 H5 YAGHQYEFFFDF, λ3 QSRDPVGFP,  ~36 nM >100 nM SEQ ID NO: 27SEQ ID NO: 70 MS-BW-31 H5 LYADADIYFDY, λ2 QSYDLSPR, ~13 +/− 9 nM >100 nMSEQ ID NO: 28 SEQ ID NO: 71 MS-BW-32 H1A TKYVGSEDV, λ2 QSYDFSHYFF,  ~92nM >100 nM SEQ ID NO: 29 SEQ ID NO: 72 MS-BW-36 H5 YRYPHMFDF, λ3QSYDLRYSH,  ~42 nM  ~75 nM SEQ ID NO: 30 SEQ ID NO: 73 MS-BW-37 H5LFAGLELYFDY, λ2 QSYDLRNR,  10 +/− 9 nM >100 nM SEQ ID NO: 31SEQ ID NO: 74 MS-BW-38 H3 GGFFNMDY, λ2 QSYDFTYGS, ~353 nM >100 nMSEQ ID NO: 32 SEQ ID NO: 75 MS-BW-39 H1A GYIPYHLFDY, κ3 QQFNDSPY, ~108nM >100 nM SEQ ID NO: 33 SEQ ID NO: 76 MS-BW-54 H5 YYGFEYDLLFDN, λ2QSYDISGYP,   9 +/− 1 nM    7 nM SEQ ID NO: 34 SEQ ID NO: 77 MS-BW-55 H1BITYIGYDF,  λ2 QSRDLYYVYY,  ~23 nM ~100 nM SEQ ID NO: 35 SEQ ID NO: 78MS-BW-56 H1A QEWYMDY,  λ3 QSYDRSMW, ~170 nM >100 nM SEQ ID NO: 36SEQ ID NO: 79 MS-BW-57 H5 LYPEDLIYFDY, λ2 QSWDVQTDK,  ~39 nM  ~60 nMSEQ ID NO: 37 SEQ ID NO: 80 MS-BW-58 H6 WMTPPGHYYGYTFDV, λ3 QSWDPSHYY,~138 nM not tested SEQ ID NO: 38 SEQ ID NO: 81 MS-BW-59 H5 LRVHDYAMYFDL,λ2 QSYDIMPER,  ~15 nM 30 +/− 5 nM SEQ ID NO: 39 SEQ ID NO: 82 MS-BW-60H5 FVSYNGSVPYFDY, λ2 QSMDFRLMH,  ~30 nM >100 nM SEQ ID NO: 40SEQ ID NO: 83 MS-BW-61 H5 IIGDYVIFFDV, λ2 QSFDMIHPY,  ~51 nM >100 nMSEQ ID NO: 41 SEQ ID NO: 84 MS-BW-62 H5 LFTYPFLYFDV, λ2 QSDFPVM,  ~36 nM19 +/− 2 SEQ ID NO: 42 SEQ ID NO: 85 MS-BW-63 H5 ILTGHVLLFDY, λ2QSDNPYL,  ~14 nM 20 +/− 1 nM SEQ ID NO: 43 SEQ ID NO: 86 *In cases werestandard deviations are given, three independent measurements were donewith Fab from three different protein expressions/purifications.~Indicates preliminary data, in cases where measurement was done onlyonce.

Example 20 Increasing the Affinity of Selected Anti-Rat TIMP-1Antibodies

Affinity maturation was applied to increase the affinity of monovalentanti-rat TIMP-1 Fab fragments to the sub-nanomolar range. No clearsequence homology could be identified among the light chain CDR3sequences of the selected antibody fragments, indicating that an optimallight chain CDR3 sequence was probably not present or had not beenselected from the original HuCAL®-Fab 1 library. We therefore startedwith modification of LCDR3 to increase the affinity of Fabs.

Two affinity maturation libraries based on MS-BW-14, -17, and -54 clonedinto phage display vector pMORPH® 18 were created. In library 1, onlyLCDR3 was diversified using TRIM technology, as described in Virnekäs etal., Nucl. Acids. Res. 22, 5600-07, 1994; Knappik et al., J. Mol. Biol.296, 57-86, 2000. In library 2, LCDR1, LCDR2, and LCDR3 were diversifiedsimultaneously using the TRIM technology, while the connecting frameworkregions were kept constant. In both cases, phage antibody librariescomprising 3×10⁸ different clones were obtained. Both libraries weremixed and used as input for a modified AutoPan® procedure. To selectantibodies having an increased affinity to rat TIMP-1, solution panningusing limiting amounts of biotinylated antigen and stringent washingconditions were applied.

Antibody-off-rates were ranked by BIAcore™ using crude E. coli extracts.Clones with slower off rate than parental clones MS-BW-14, -17, or -54were subjected to expression and purification in 1-liter scale. PurifiedFab were analyzed in BIAcore™ and rat protease assays (Table 6).MS-BW-17-1 (K_(d) 0.8 nM, IC₅₀ 1.6 nM), MS-BW-17-2 (K_(d) 1.3 nM, IC₅₀1.1 nM), and MS-BW-17-3 (K_(d) 1.9 nM, IC₅₀ 3 nM) were derived fromaffinity maturation library 1 having an optimized LCDR3 sequence,whereas MS-BW-54-1 (K_(d) 2 nM, IC₅₀ 3 nM) was derived from affinitymaturation library 2 having an optimized LCDR1, -2, and -3 sequence(Table 9).

TABLE 9 Overview and sequence comparison of affinity matured Fab fragmentsagainst rat TIMP-1. Sequence changes compared to parentalFab fragments (bold) are italicized. VL Clone HCDR1 VH (MS- Frame-sequence HCDR2 sequence HCDR3 sequence Frame- BW-) work (SEQ ID NO:)(SEQ ID NO:) (SEQ ID NO:) work 14 VH1A GGTFSSYAIS GIIPIFGTANYAQKFQGWSDQSYHYYWHPYFDV VL1 (366) (368) (370) 17 VH5  GYSFTSYWIGIIYPGDSDTRYSPSFQG LTNYFDSIYYDH VL2 (367) (369) (18) 54 VH5  GYSFTSYWIGIIYPGDSDTRYSPSFQG YYGFEYDLLFDN VL2 (367) (369) (34) 17-1 VH5  GYSFTSYWIGIIYPGDSDTRYSPSFQG LTNYFDSIYYDH VL2 (367) (369) (18) 17-2 VH5  GYSFTSYWIGIIYPGDSDTRYSPSFQG LTNYFDSIYYDH VL2 (367) (369) (18) 17-3 VH5  GYSFTSYWIGIIYPGDSDTRYSPSFQG LTNYFDSIYYDH VL2 (367) (369) (18) 54-1 VH5  GYSFTSYWIGIIYPGDSDTRYSPSFQG YYGFEYDLLFDN VL2 (367) (369) (34) VH Monov. K_(D)Clone LCDR1 LCDR2 LCDR3 to rat IC₅₀ in rat (MS- sequence sequencesequence TIMP-1 protease BW-) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)(nM) assay (nM) 14 SGSSSNIGSNYVS LMIYDNNQRPS QSWDLEPY 10 +/− 5 14 +/− 3(371) (373) (59) 17 TGTSSDVGGYNYVS LMIYDVSNRPS QSYDPSHPSK 13 +/− 311 +/− 3 (363) (374)  (61) 54 TGTSSDVGGYNYVS LMIYDVSNRPS QSYDISGYP 9 +/− 1 7 (363) (374) (77) 17-1 TGTSSDVGGYNYVS LMIYDVSNRPS QAFDVAPNGK0.8 1.6 (363) (374)  (376) 17-2 TGTSSDVGGYNYVS LMIYDVSNRPS QAFAVMPNVE1.3 1.1 (363) (374)  (377) 17-3 TGTSSDVGGYNYVS LMIYDVSNRPS QSFTVSPGAD1.9 3 (363) (374)  (378) 54-1 TGTSSDLGGYNYVS LMIYAGNNRPS QAYDSSGYP 2 3(372) (375) (379)

The improvement gained by these different one-step maturation strategieswas up to a factor of 16.3 with regard to affinity and 10 with regard tofunctional activity in the protease assay.

Example 21 Conversion of Anti-TIMP-1 Fab Fragments into Human IgG₁Molecules for Use in the Rat Model of Chronic CarbonTetrachloride-Induced Liver Fibrosis

Anti-TIMP-1 Fab fragments were converted into human IgG1 molecules tocreate antibody molecules with prolonged in vivo half-lives for the usein the rat model of chronic carbon tetrachloride-induced liver fibrosis.This was done by cloning the heavy and light chain variable regions ofthe Fab into two separate vectors for mammalian IgG₁ expression (Krebset al., 2001)

Anti-rat TIMP-1 clone MS-BW-14 was chosen for the first in vivo study,and IgG₁ protein was produced by transient expression. Anti-human TIMP-1clone MS-BW-3 was selected as a negative control IgG₁ and was alsoproduced by transient expression. Purified IgG₁ proteins MS-BW-14 andMS-BW-3 were subjected to quality control in BIAcore™ and rat TIMP-1/ratMMP-13 assays. Bivalent affinity for rat TIMP-1 measured in BIAcore™(chip density 500 RU, fitting model for bivalent analyte) is 0.2 nM forMS-BW-14, compared to 13 nM for the corresponding monovalent Fabfragment. This increase in affinity for the IgG₁ is due to the avidityeffects caused by binding of bivalent IgG₁ to immobilized rat TIMP-1protein on the BIAcore™ chip. As expected, the negative control IgG₁MS-BW-3 showed no binding to rat TIMP-1 but bound to human TIMP-1 with abivalent affinity of approximately 0.4 nM.

FIG. 12 shows the activity of MS-BW-14 Fab and IgG₁ and MS-BW-3 IgG₁ ina rat TIMP-1/rat MMP-13 assay. The IC₅₀ of MS-BW-14 Fab and IgG₁ arenearly identical. The avidity effect seen in BIAcore™ does not occur inthis assay because, in contrast to the BIAcore™ experiment, this assayis based on a monovalent interaction in solution between TIMP-1 and theIgG₁. As expected, MS-BW-3 has no effect on rat TIMP-1 binding to ratMMP-13 and thus is a suitable negative control for a rat in vivo study.

Affinity matured clone MS-BW-17-1 was then converted from a monovalentFab fragment to a bivalent IgG₁. Protein was produced by stabletransfection. Purified protein was subjected to quality control inBIAcore™ and rat TIMP-1/rat MMP-13 assays (FIG. 13). In BIAcore™ anincreased bivalent affinity (avidity) of 0.04 nM for IgG₁ compared to0.8 nM for monovalent Fab fragment was seen, whereas the activity in therat TIMP-1/rat MMP-13 assay was comparable for IgG₁ and Fab as expected.

Example 22 Cross-Reactivity of Anti-Rat TIMP-1 IgG₁ MS-BW-17-1 withMouse TIMP-1

Species cross-reactivity of MS-BW-17-1 IgG₁ and Fab with mouse TIMP-1was determined by BIAcore™ to investigate the feasibility of alternativein vivo models that use mice instead of rats. Although MS-BW-17-1clearly bound to mouse TIMP-1 immobilized to the chip surface, theaffinity of both Fab (180 nM) and IgG₁ (9 nM) was 225-fold weaker thanthe affinity to rat TIMP-1. As the interaction between mouse TIMP-1 andBW-17-1 IgG₁ in serum is most likely monovalent, the affinity of BW-17-1Fab probably reflects the “real” affinity of this interaction.Therefore, the Fab affinity value should be considered when calculatingthe feasibility of using BW-17-1 IgG₁ in a mouse in vivo study.

Example 23 Effect of Timp-1 Antibody on the Development ofBleomycin-Induced Pulmonary Fibrosis

The following example demonstrates the ability of a human anti-ratTimp-1 antibody (BW17.1) to prevent fibrotic collagen deposition in ableomycin-induced rat lung fibrosis model.

Male Lewis rats (6 weeks of age) received a single intratrachealchallenge with bleomycin (0.3 mg/rat, in saline) or vehicle (saline) onday 0. Fourteen days later, animals were euthanized, the lung excised,fixed, and processed for evaluation of lung fibrosis. Lung tissuesections were cut, and quantitative assessment by image analysis of lungcollagen in lung tissue sections stained with Mason Trichrome stainperformed.

Antibody administration: A 20 mg/kg dose of human ant-rat TIMP-1antibody or control human antibody (IgG) was administered subcutaneouslyon day −1. Subsequently, a 10 mg/kg dose of human ant-rat TIMP-1antibody or control human antibody (IgG) was administered s.c. on days2, 5, 8, and 11. The following five groups of animals were studied:Saline i.t. challenge+antibody vehicle (PBS); Saline i.t.challenge+TIMP-1 antibody; Bleomycin i.t. challenge+TIMP-1 antibody;Bleomycin i.t. challenge+antibody vehicle (PBS); Bleomycin i.t.challenge+control antibody.

FIG. 14 shows the effect of the inhibitory effect of TIMP-1 antibody onbleomycin-induced lung fibrotic collagen.

Example 24 Effect of BW-14 Anti-TIMP-1 Antibody in a Rat Model withCCl₄-Induced Liver Fibrosis

Carbon tetrachloride (CCl₄) was used to induce liver fibrosis asdescribed in Example 9. A single intravenous dose of 3 mg/kg BW-14 orcontrol antibody BW-3, respectively, was administered on day 19. At thistime, total liver collagen (hydroxyproline determined according toProckop and Udenfried) is already significantly increased by CCl₄, andfibrotic collagen rapidly accumulates during the following weeks. Therats were sacrificed on day 28. The treatment groups were: no CCl₄+control antibody BW 3 (n=10 rats), CCl₄+ control antibody BW 3 (n=20rats), and CCl₄+BW 14 (n=20 rats).

The effect of control vs. TIMP-1 antibody as reflected in morphometricmeasurements of fibrous collagen (Sirius Red stained area as percentageof the total field) is shown in FIG. 15. Comparison of both controlantibody treated groups shows that CCl₄ caused an approximatelythree-fold increase in collagen area. BW-14 antibody treatment reducedthe pathological collagen increment by 26%. The lower fibrous collagenvalue of the CCl₄+BW-14 group compared to the CCl₄+BW-3 group wasstatistically significant (p<0.05, Kolmogorow-Smirnow test).

REFERENCES

-   Ausubel et al. (1998) Current Protocols in Molecular Biology. Wiley,    New York, USA.-   Better et al., (1988) Escherichia coli secretion of an active    chimeric antibody fragment. Science 240, 1041.-   Bruggeman et al., (1996) Phage antibodies against an unstable    hapten: oxygen sensitive reduced flavin. FEBS Lett. 388, 242.-   Butler et al., (1999) Human tissue inhibitor of metalloproteinases 3    interacts with both the N- and C-terminal domains of gelatinases A    and B. Regulation by polyanions. J Biol. Chem. 274, 10846.-   Gomis-Ruth et al., (1996). Mechanism of inhibition of the human    matrix metalloproteinase stromelysin-1 by TIMP-1. Nature. 389, 77.-   Griffiths, A. D. and Duncan, A. R. (1998) Strategies for selection    of antibodies by phage display. Curr. Opin. Biotechnol. 9, 102.-   Hoogenboom, H. R. and Winter, G. (1992). By-passing immunisation.    Human antibodies from synthetic repertoires of germline VH gene    segments rearranged in vitro. J. Mol. Biol. 227, 381.-   Iredale et al., (1996) Tissue inhibitor of metalloproteinase-1    messenger RNA expression is enhanced relative to interstitial    collagenase messenger RNA in experimental liver injury and fibrosis.    Hepatology. 24, 176.-   Knappik et al., (2000) Fully synthetic human combinatorial antibody    libraries (HuCAL) based on modular consensus frameworks and CDRs    diversified with trinucleotides. J. Mol. Biol. 296, 55.-   Krebs et al., (2001) High-throughput generation and engineering of    recombinant human antibodies. J Immunol Methods. 254, 67.-   Lowman, H. B. (1997) Bacteriophage display and discovery of peptide    leads for drug development. Annu Rev. Biophys. Biomol. Struct. 26,    401.-   McCafferty et al., (1990) Phage antibodies: filamentous phage    displaying antibody variable domains. Nature 348, 552.-   Meng et al., (1999) Residue 2 of TIMP-1 is a major determinant of    affinity and specificity for matrix metalloproteinases but effects    of substitutions do not correlate with those of the corresponding    P1′ residue of substrate. J Biol. Chem. 274, 10184.-   Meulemans et al., (1994) Selection of phage-displayed antibodies    specific for a cytoskeletal antigen by competitive elution with a    monoclonal antibody. J. Mol. Biol. 244, 353.-   Miyazaki et al., (1999) Changes in the specificity of antibodies by    site-specific mutagenesis followed by random mutagenesis. Protein    Eng. 12, 407.-   Sheets et al., (1998) Efficient construction of a large nonimmune    phage antibody library: The production of high-affinity human    single-chain antibodies to protein antigens. Proc. Natl. Acad. Sci.    U.S.A. 95, 6157.-   Skerra, A. and Pliickthun, A. (1988) Assembly of a functional    immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038.-   Smith, G. P. (1985) Filamentous fusion phage: novel expression    vectors that display cloned antigens on the virion surface. Science    228, 1315.-   Smith, G. P. and Petrenko, V. A. (1997) Phage display. Chem. Rev.    97, 391.-   Stausbøl-Gron et al., (1996) A model phage display subtraction    method with potential for analysis of differential gene expression.    FEBS Lett. 391, 71.-   Virnekäs et al. (1994) Trinucleotide phosphoramidites: ideal    reagents for the synthesis of mixed oligonucleotides for random    mutagenesis. Nucl. Acids Res. 22, 5600.

1. A method of ameliorating symptoms of a disorder in which TIMP-1 iselevated, comprising the step of: administering to a patient having thedisorder an effective amount of an antibody, wherein the antibody bindsto a tissue inhibitor of metalloprotease-1 (TIMP-1); neutralizes amatrix metalloprotease (MMP)-inhibiting activity of the TIMP-1; andcomprises: a VHCDR1 region comprising an amino acid sequence as setforth in SEQ ID NO:356; a VHCDR2 region comprising an amino acidsequence as set forth in SEQ ID NO:358; a VHCDR3 region comprising anamino acid sequence as set forth in SEQ ID NO:3; a VLCDR1 regioncomprising an amino acid sequence as set forth in SEQ ID NO:363; aVLCDR2 region comprising an amino acid sequence as set forth in SEQ IDNO:364; and a VLCDR3 region comprising an amino acid sequence as setforth in SEQ ID NO:365, wherein the disorder is liver fibrosis.
 2. Themethod of claim 1, wherein the MMP is human MMP-1.
 3. The method ofclaim 1, wherein the TIMP-1 is a human TIMP-1.
 4. A method ofameliorating symptoms of a disorder in which TIMP-1 is elevated,comprising the step of: administering to a patient having the disorderan effective amount of a composition comprising a human purifiedantibody which (1) binds to a TIMP-1; (2) neutralizes an MMP-inhibitingactivity of the TIMP-1; and (3) comprises a VHCDR1 region comprising anamino acid sequence as set forth in SEQ ID NO:356; a VHCDR2 regioncomprising an amino acid sequence as set forth in SEQ ID NO:358; aVHCDR3 region comprising an amino acid sequence as set forth in SEQ IDNO:3; a VLCDR1 region comprising an amino acid sequence as set forth inSEQ ID NO:363; a VLCDR2 region comprising an amino acid sequence as setforth in SEQ ID NO:364; and a VLCDR3 region comprising an amino acidsequence as set forth in SEQ ID NO:365; and a pharmaceuticallyacceptable carrier, wherein the disorder is liver fibrosis.
 5. Themethod of claim 4, wherein the MMP is human MMP-1.
 6. The method ofclaim 4, wherein the TIMP-1 is a human TIMP-1.